Laboratory Manual For Diagnosis of Sexually Transmitted and Reproductive Tract Infections
Laboratory Manual For Diagnosis of Sexually Transmitted and Reproductive Tract Infections
Laboratory Manual For Diagnosis of Sexually Transmitted and Reproductive Tract Infections
Sexually Transmitted Infections (STIs) and Reproductive Tract Infections (RTIs) are diseases of
major global concern. About 6% of Indian population is reported to be having STIs. In addition to
having high levels of morbidity, they also facilitate transmission of HIV infection. Thus control of
STIs goes hand in hand with control of HIV/AIDS.
This manual aims to bring in standard testing practices among laboratories that serve health
facilities involved in managing STIs and RTIs. While generic procedures such as staining,
microscopy and culture have been dealt with in detail, procedures that employ specific
manufacturer defined kits have been left to the laboratories to follow the respective protocols.
An introduction to quality system essentials and quality control principles has also been
included in the manual to sensitize the readers on the importance of quality assurance and
quality management system, which is very much the need of the hour.
Sexually Transmitted Infections (STIs) are the most common infectious diseases worldwide,
with over 350 million new cases occurring each year, and have far-reaching health, social, and
economic consequences. Failure to diagnose and treat STIs at an early stage may result in
serious complications and sequelae. Since most of these infections occur in the economically
productive age group the issue assumes utmost priority.
STIs are passed from person to person primarily by sexual contact and are classified into varied
groups. Some cause mild, acute symptoms and some are life-threatening. They are caused by
many different infectious organisms and are treated in different ways. Antimicrobial resistance
of several sexually transmitted pathogens is increasing, rendering some regimens ineffective,
adding to therapeutic problems.
Apart from being serious diseases in their own right, STIs enhance the sexual transmission of
HIV infection. The presence of an untreated STIs (ulcerative or non-ulcerative) can increase
the risk of both acquisition and transmission of HIV by a factor of up to 10.
It has been estimated that improvement in the management of STIs can reduce the incidence of
HIV-1 infection in the general population by about 40%. STIs prevention and treatment are,
therefore, an important component in HIV prevention strategy. Correct and early detection of
STIs using standardized uniform protocols is essential at all levels in order to institute treatment
and control measures.
This manual is written with an intention to bring uniform, detailed testing protocols for STIs and
some RTIs at primary, intermediate and central laboratory levels and also to recommend sample
transport mechanism and referral network.
Page No.
A. Introduction 1
B. Stratification of laboratories 5
C. Collection and transport of specimens 9
D. Laboratory Procedures 14
1. Gonorrhoea 14
2. Chlamydial Infections 34
3. Syphilis 39
4. Chancroid 53
5. Granuloma inguinale 57
6. Bacterial vaginosis 60
7. Candidiasis 64
8. Trichomoniasis 70
9. Herpes simplex virus infections 77
10. Human papilloma virus infection 81
11. Hepatitis B 83
12. Hepatitis C 84
E. Biosafety 85
F. Washing and sterilization of Glassware 89
G. Maintenance of equipment 92
H. Quality assurance, Quality control, Quality assurance programme 94
I. Registration and record keeping 97
Appendix:
Annexure-I - Microscopy 99
Annexure- II - Culture Media 105
Annexure-III - Guidelines for sample and patient transfer 120
Annexure-IV - Requirements for STI/RTI laboratory at RSTRRL/SRC 122
Annexure-V - Bacteriology procedures 129
Annexure-VI - Urine examination 143
More than 30 bacterial, viral and parasitic pathogens are transmissible sexually and constitute a
group of infections referred to as STIs. Most common are Gonorrhoea, Chlamydial infection,
Syphilis, Chancroid, Human Immunodeficiency Virus (HIV) infections, Herpes progenitalis
(Herpes simplex virus infection), Trichomoniasis, Candidiasis, etc. Although some of the
pathogens can be acquired through routes other than sexual transmission, epidemiologically,
sexual contact is more important for their transmission from one person to another.
The infected individual acts as a source of infection for other persons. The infection may occur
during heterosexual or homosexual activities. STI patients may give a history of multiple sex
partners but contact with even a single infected sex partner may be enough to contract infection.
STIs, remain an important focus area for global public health. Public health strategies for STIs
control include promotion of safer sexual behaviour and provision of condoms (primary
prevention), as well as early and efficacious management of patients with STIs, using either
syndromic or etiological management approaches.
Reproductive Tract Infections (RTIs) include sexually transmitted infections as well as infections
of the reproductive tract that are not sexually transmitted. RTIs including STIs and HIV/AIDS are
being increasingly recognized as a serious public health problem. RTIs cause suffering both to
men and women. RTIs present commonly with vaginal and urethral discharges, pain or burning
with urination, itching or irritation of the genitals, sores, blisters or swellings on the genitals and
pelvic pain. Adolescents too are vulnerable to RTIs due to their ignorance of risk factors,
inadequate accessibility to services and social powerhouses. A number of studies have been
initiated to cover the epidemiological, clinical and diagnostic dimensions of RTIs.
Based on the types of lesion and the presenting symptoms, STIs can be grouped as those with
a) ulcers b) discharge c) pain and swelling and d) others. This classification is useful to facilitate
sample collection and syndromic treatment. *
* Syndromic management is based on the identification of consistent groups of symptoms and easily recognized
signs (syndromes), and the provision of treatment that will deal with the majority or most serious organisms
responsible for producing a syndrome.
Recognizing the need to provide prompt diagnosis for initiation of treatment and also keeping in
mind that many confirmatory tests require specialised equipment, expertise or environment,
laboratories have been classified into three levels. A referral network system from periphery to
Intermediate and Central laboratory is available.
3. Central/Regional Reference Laboratory: These laboratories are well- equipped with state of
the art equipment and highly trained staff to perform more sophisticated confirmatory tests
apart from the tests suggested at primary and intermediate laboratories. They undertake
diagnosis, surveillance and research in sexually transmitted infections in their areas. These
laboratories also are expected to ensure quality of tests performed in the intermediate
laboratories/state reference laboratories linked to them, to train the laboratory staff and
participate in evaluations /studies organized by the apex laboratory. Regional STI referral
laboratories, well-equipped medical colleges and selected BioMedical Research institutes
constitute central laboratories.
Taking into consideration the resources available in the country, the tests recommended at the
different levels of the health care system are shown in the following Table.
Culture - + + +
Identification of organism - + + +
Neisseria
gonorrhoeae β-lactamase test - ± + +
Antigen detection
1)Direct fluorescent antibody - - + +
technique (DFA)
2)ELISA - - + +
Chlamydia
trachomatis Culture - - ± +
VDRL (Qualitative) - + + +
Treponema
pallidum VDRL (Quantitative) - + + +
TPHA - + + +
FTA-Abs test - - ± +
Molecular identification - - ± +
Culture - ± + +
Antigen detection
1) Immuno-fluorescent staining - ± + +
Herpes
Simplex 2)ELISA - ± + +
Virus-1&2
Antibody detection
Anti- HSV IgM (type specific - ± + +
glycoprotein) G[gG]
Culture - - ± +
Culture - + + +
Culture - ± + +
Human
Papilloma Nucleic Acid Amplification - - + +
Viruses
Immunochromatography
HBs Ag detection + + + +
Anti-HCV antibody
Hepatitis C detection - ± + +
virus
Nucleic Acid Amplification Tests - ± ± +
Collection of an adequate specimen from the proper site from the right patient with symptoms
is essential for correct diagnosis (Right patient, right test and right sample!). The site for
collection of the specimen will depend on the clinical symptoms. It is essential to follow
Standard precautions at all times during specimen collection, storage, testing, transportation
and disposal of bio-hazardous wastes. Standard precautions are meant to reduce the risk of
transmission of blood borne and other pathogens from both recognized and unrecognized sources.
The anogenital specimen in males can be collected by the medical officer himself or by the
technician. In females the specimen should, preferably, be collected by a lady medical officer, a
nurse or a lady health worker only.
Urethral swab Should be done after atleast one hour of voiding urine. Express
urethral exudates when patients have discharge, collect with a sterile
swab.
If there is no discharge, compress the meatus vertically to open the
distal urethra and insert a thin, water-moistened swab (calcium
alginate or Dacron) with flexible wire slowly (3 cm to 4 cm in males or 1
cm to 2 cm in females), rotate slowly and withdraw gently. Do not use
calcium alginate swabs for viral cultures.
Epididymis Use a needle and syringe to aspirate material from the epididymis and
collect in a tube.
Cervical swab Insert a speculum into the vagina to view the cervix. Wipe the cervix
clean of vaginal secretion and mucus.
Insert a swab 1 cm to 3 cm into the endocervical canal and rotate for 10
s to 30 s to allow absorption of exudates
In cases of suspected coinfections of N. gonorrhoeae and Chlamydia
trachomatis, the cervical specimen for N gonorrhoeae detection
should be taken before the specimen for C. trachomatis, because N.
gonorrhoeae is present in the mucus from the endo-cervix and C.
trachomatis is present in the cervical epithelial cells. A small brush on a
wire (cytobrush) is used to collect specimens in females in cases of
C.trachomatis infections.
Vaginal swab Collect pooled vaginal secretions, if present. Vaginal wash specimens*
are most preferred and acceptable from prepubertal girls. If not
possible, rub a sterile cotton swab against the posterior vaginal wall
and allow the swab to absorb the specimen.
Transport of specimens
Specimens and associated materials must be packaged and transported in a suitable manner, to:
} protect the safety of everyone required to handle the specimens and package
} ensure that the material is maintained under suitable conditions and is stable to perform the
required test.
Packing of specimens:
The system consists of three layers as follows
1. Primary receptacle. It is a labeled primary watertight, leak-proof receptacle containing the
specimen. The receptacle is wrapped in enough absorbent material to absorb all fluid in case of
breakage.
2. Secondary receptacle. It is a second durable, watertight, leak-proof receptacle to enclose
and protect the primary receptacle(s). Several wrapped primary receptacles may be placed
in one secondary receptacle. Sufficient additional absorbent material must be used to
cushion multiple primary receptacles. Figure 1 -
Specimen data forms, letters and other Triple packaging system
types of information that identify or and Bio-hazard symbol
describe the specimen test and also
identify the shipper and receiver should
be taped to the outside of the
secondary receptacle, preferably in a
zip pouch.
3. Outer shipping package. The secondary
receptacle is placed in an outer shipping
package which protects it and its
Ziploc plastic bags may also be used as leak-proof containers if suitable boxes are not available.
Packed specimens should be sent to the nearest referral laboratory for further tests.
* VTM (Viral Transportation Medium) is recommended for transportation of samples for viral culture at 2 to 8 OC. Samples should be
transported within 48 hours.
1. GONORRHOEA
Gonorrhoea is caused by aerobic gram-negative diplococci, Neisseria gonorrhoeae.
Demonstration of intracellular gonococci in Gram stained smears made from urethral
discharges have very good sensitivity and specificity at primary care level. In cases with cervical
oro-pharyngeal and rectal discharges, culture will have to be performed as microscopy has very
poor sensitivity and specificity.
1.2 Microscopy
Purpose of microscopy is to stain the smears by Grams method and look for pus cells, intra-
cellular Gram-negative diplococci suggestive of gonococci.
1. With one swab prepare a smear on a clean grease free slide (Annexure-I)
2. Allow the smear to dry in the air. Figure 2 - Gram Stain of N. gonorrhoeae
3. Heat fix the smear (Annexure-I)
4. Stain with Gram stain (Annexure-I).
5. Examine the slide using light microscope.
First focus under 10x objective and then put a
drop of liquid paraffin (immersion oil) on the
smear and examine under 100x objective.
Push the condenser up and open the iris
diaphragm so that maximum light passes
through the slide.
Reading: Examine the smear for epithelial cells, polymorphonuclear leucocytes (pus cells),
organisms and their location- whether extracellular or intracellular. The gonococci are
intracellular, bean shaped and are usually arranged in pairs, 0.8µm x 0.6µm in size. They are
Gram stain
Material required:
i) Oxidase reagent (tetra methyl para- phenylenediamine dihydrochloride).
ii) Distilled water.
iii) Filter paper strips.
iv) Glass slides / wooden applicator stick
Method I:
i) Prepare a 1% fresh solution of oxidase reagent by dissolving 10 mg of reagent in 1 ml of
distilled water.
ii) Put a drop of the reagent on the colony to be tested.
iii) Immediate development of a purple colour indicates a positive test.
Method II:
i) Moisten a filter paper strip with 2-3 drops of oxidase reagent.
ii) Pick one colony with the wooden applicator stick or tip of glass slide or tooth pick or
platinum loop and rub on filter paper.
Method III:
i) Cut Whatman filter paper into small strips.
ii) Pour 1% fresh solution of oxidase reagent on to the strips in a big petri dish.
iii) Dry them in hot air oven.
iv) Store the strips in a brown bottle in refrigerator.
Reading:
i) Dark deep purple colour in 5 – 10 sec – positive test.
ii) Deep purple colour in 10 – 60 sec. – Delayed positive test.
iii) No colour or colour development after 60 sec. – negative test.
Plate method:
Perform by placing one drop of 30% w/v hydrogen peroxide solution on chocolate agar. An
immediate and abundant production of bubbles indicates a positive test. Delayed or weak
bubbling indicates a negative test.
N. gonorrhoeae (100%) and some other commensal Neisseria give a positive reaction. More than 98%
of N. meningitidis are negative in this test. A negative test means that the isolate is not a gonococcus.
Acidometric test for the presence of beta-lactamase, may be conveniently performed along
with RCUT. β-lactamase, an extracellular enzyme produced by many strains of bacteria,
specifically hydrolyses the amide bond in the β-lactam ring of penicillin analogues, rendering
the antibiotic inactive. Penicillinoic acid is formed with a resulting colour change. A test for
β-lactamase from N. gonorrhoeae can be detected by the change in colour of phenol red pH
indicator (red to yellow) when penicillin converts to penicillinoic acid.
Ampicillin, is more stable and more sensitive to TEM β-lactamase, and is used instead of
penicillin G for the test. The test can thus be used both for identification and testing for
β-lactamase production.
Materials required:
1. Buffered salt indicator solution
2. Growth medium, e.g., non-selective chocolate agar plates
3. Carbohydrate solutions
10% glucose (G)
10% lactose (L)
10% sucrose (S)
10% maltose (M)
4. Ampicillin solution for β-lactamase test
Take a microtitre plate and mark its 6 wells as C (control), G (Glucose), L (Lactose), M (Maltose), S
(Sucrose) and P’ase (penicillinase). Add 25 µl of 10% sterile sugar solution to G, L, M, S wells and
25 µl of ampicillin solution (200 mg/mL) to P’ase well. The first well without any sugar will serve
as a control. Add 100µl (4 drops) of bacterial suspension to each of the six wells.
Read after 2 to 4 hours of incubation at 35OC - 37OC in air (not in CO2). It is recommended that the
β-lactamase reaction is examined again after 24 hours as slow β-lactamase reactions occur with
occasional strains.
The following control strains are set up simultaneously with each test run.
Neisseria gonorrhoeae WHO E or WHO O: β-lactamase POSITIVE
Neisseria gonorrhoeae WHO C or WHO K: β-lactamase NEGATIVE
Note:
1. If N.gonorrhoeae is suspected and a doubtful result is obtained with the rapid carbohydrate
utilization test, check the purity of the culture and send it to the central laboratory for confirmation.
2. The RCUT should not be performed in glass tubes.
Results:
The results of carbohydrate utilization and β-lactamase production are recorded as follows:
Control tube/well = Red.
Yellow colour = Positive reaction
Orange Red colour = Negative reaction
N.gonorrhoeae utilizes glucose only but not maltose and lactose, so only the tube containing
glucose should have a colour change.
Interpretation of sugar utilization test
+ - - - N.gonorrhoeae
+ + - - N.meningitidis
+ + + - N.lactamica
- - - - Refer to reference laboratory
for further confirmation
Method:
a) Disk method:
I) Wet a nitrocefin disk with a drop of distilled water.
ii) Pick 5 colonies with sterilized platinum loop and rub on the disk
Reading:
Red colour within 1 minute – positive test.
Original yellow colour – negative test.
b) Slide Method:
i) Prepare nitrocefin solution by mixing freeze dried powder with the dilution buffer.
ii) Put one drop of nitrocefin solution on a piece of filter paper over a slide.
iii) Pick up 10-20 colonies of N.gonorrhoeae with a sterile platinum loop.
iv) Rub the colonies with the reagent on the filter paper.
Reading:
Red colour in 30 seconds – Positive test.
Yellow colour – Negative test.
Patient Populations
} STI patients (individuals presenting for treatment with signs and symptoms)
} Screening/case finding (individuals who are mostly asymptomatic for STI, about are tested
for gonorrhoea as part of an early detection programme)
Antibiotics in the ‘additional’ group include those that are used for the treatment of gonorrhoea
in some parts of the world.
Core Group
1. Penicillins used as the representative antibiotic. Results apply to ampicillin and
amoxycillin.
2. Quinolones ciprofloxacin is currently recommended for testing as the representative
quinolone. Results of ciprofloxacin testing may be applied to other quinolone
agents.
3. Ceftriaxone used as a representative of injectable third generation cephalosporins.
4. Cefixime used as a representative of oral third generation cephalosporins.
5. Tetracyclines used only to detect strains with high-level plasmid mediated resistance to
tetracycline-referred to as TRNG strains.
6. Spectinomycin useful against penicillin and ceftriaxone resistant isolates.
7. Azithromycin recommended as part of a dual treatment strategy.
Additional group
Other cephalosporins (cefpodoxime), aminoglycosides.
Concomitant with diffusion of the drug, the bacteria that were inoculated and that are not
inhibited by the concentration of the antimicrobial agent continue to multiply until a lawn of
growth is visible. In areas where the concentration of drug is inhibitory, no growth occurs,
forming a zone of inhibition around each disk.
Inoculum: pick up few colonies from an overnight (20 -24 hrs) chocolate agar plate; make a
direct suspension equivalent of 0.5 MacFarland standard; prepare in Mueller Hinton Broth or
0.9% phosphate buffered saline, pH7.0.
Materials
Chocolate Agar with growth supplement
Phosphate buffered saline (Annexure-II)
Sterile swabs
Penicillin 10 IU.
Tetracycline 30 µg.
Ceftriaxone 30 µg.
Spectinomycin 100 µg.
Ciprofloxacin 5 µg.
Cefixime 5 µg.
Azithromycin 15µg
Procedure
1. Allow disks to come to room temperature before opening the container.
2. Prepare a suspension of the test organism in sterile saline equivalent to a 0.5 McFarland
standard using isolated colonies.
3. Using a sterile cotton swab, inoculate the organism on agar
4. Using forceps or a disk dispenser, apply the appropriate antimicrobial disks onto the agar.
Place the disks with an equal distance apart from each other and put not more than 6 disks
on a 90 mm diameter plate.
O
5. Incubate plates at 35 C for 18-24 hrs.
Measure the diameter of the clear zone by placing the ruler on the inoculated surface of the
petridish (The side on which the code word for the antibiotic is written).The size of the zone
indicates the sensitivity of gonococcus (resistant, intermediate sensitive or sensitive).
Zone size in mm
Drug
Resistant Intermediate Sensitive Sensitive
Penicillin ≤ 26 27-46 ≥47
Tetracycline ≤30 31-37 ≥38
Spectinomycin ≤14 15-17 ≥18
Ceftriaxone - - ≥35
Cefixime - - ≥31
Ciprofloxacin ≤27 28-40 ≥41
Azithromycin ≤30 - -
Medium:
Chocolate agar comprising Columbia agar base are suitable. 20 ml agar is dispensed into a 90
mm Petri dish so that depth of agar in each plate is approximately 4 mm.
The plates are stored in an inverted position in the refrigerator before use and are used within 7
days of pouring.
O
On the day of test, the plates are dried by inverting them without the lid in an incubator at 35 C -
O
36.5 C for 45 minutes to an hour.
Inoculation of plates:
1. Mix the inoculum using a Pasteur pipette at least 10 times and check there are no lumps left
in the suspension.
2. Flood the dried chocolate gar plate and remove the excess inoculum.
3. Allow the plate to dry at room temperature. This may take 10-15 minutes. Plates must not be
left longer than 15 minutes after the inoculum has dried.
4. Apply antibiotic discs using sterile forceps. Ensure that discs are applied evenly but do not
reapply discs after initial contact with the agar.
5. Low concentration discs are recommended in this method. Upto 6 discs can be applied on a
single 90 mm diameter plate.
O O
6. Incubate the plates at 35 C - 36 C in air containing 5% CO2 and 70 to 80% humidity for 18
hours.
Procedure:
1. Sample colony with a straight wire
2. Prepare a suspension in 2.5 ml normal saline.
3. Inoculate a pre-dried chocolate agar plate.
4. Distribute the inoculum by rocking.
5. Remove excess inoculum.
6. Dry the plate at room temperature.
7. Load the plate with antibiotic discs.
8. Incubate for 18 hours.
9. Measure the annular radii.
10. Interpret zone sizes.
Cefpodoxime 10 µg – ≤ 12 > 12
1.
The detection of beta-lactamase production defines a gonococcus as a PPNG and as
resistant to the penicillins. Penicillin disc testing of PPNG is optional, usually no zone of
inhibition is present.
2.
Quinolone disc testing is performed using a combination of both ciprofloxacin 1 μg (a
fluoroquinolone) and nalidixic acid 30 μg (a quinolone) discs. Susceptibility to nalidixic acid is
not reported but is used in determining susceptibility to ciprofloxacin. Categories of
susceptibility to ciprofloxacin are defined as follows:
} Susceptible to ciprofloxacin.
The annular radius of the inhibitory zone around both nalidixic acid 30 μg and
ciprofloxacin 1 μg discs is >6 mm.
} Less susceptible to ciprofloxacin
The annular radius of the inhibitory zone around a nalidixic acid 30 μg disc is < 6 mm (usually
1.8.2 MIC testing can be performed by E test or agar dilution method depending on facilities
available in the laboratory.
Media:
Check the approximate depth of agar and weight of agar plates. Test the control strains and
check that the zone sizes are within the acceptable range and make sure that a good lawn
growth is obtained.
Inoculum:
Bacterial material must be seen on the tip of the straight wire. After overnight incubation there
must be confluent growth on the sensitivity plate.
Initially, reference strains should be tested for 20 consecutive days to ensure that laboratory
procedure followed is correct and reproducible consistently. Subsequently weekly quality
controls should be run. In addition, whenever there is a change in the lot no of disks, quality
checks should be done. If there is a change in the manufacturer of disks,QC should be done for
ten consecutive days before being put to regular use.
Clinical isolates should be sent to WHO Regional Reference Laboratory for Gonococal
Antimicrobial Surveillance Programme (GASP) at Vardhman Mahavir Medical College and
Safdarjung Hospital for confirmation of antimicrobial susceptibility testing results.
REFERENCE STRAINS
Table. MIC and category of sensitivity of the 2008 WHO Neisseria gonorrhoeae reference
strains panel, intended for global quality assurance and quality control of antimicrobial
resistance testing and surveillance.
WHO WHO WHO WHO WHO WHO WHO WHO
Characteristics
F G K L M N O P
Cefuroxime S I R R I I I I
a
(0.064-12) (0.064) (0.5) (12) (8) (0.5) (0.25) (1) (0.125)
Ertapenem
d
S S NSc Nsc S S S S
(0.004-0.125)
a
(0.004) (0.008) (0.125) (0.064) (0.012) (0.008) (0.032) (0.008)
Erythromycin S I I I I S I R
(0.5-4)
a
(0.5) (1) (1) (2) (1) (0.5) (1) (4)
Azithromycin S S S I S S S R
(0.125-2)
a
(0.125) (0.25) (0.25) (0.5) (0.25) (0.125) (0.25) (2)
LLRe HLRe
e
Ciprofloxacin S HLR R R S S
a
(0.004->32) (0.004) (0.125) (>32) (>32) (2) (4) (0.008) (0.004)
Spectinomycin S S S S S S R S
a
(16->1024) (32) (16) (16) (16) (16) (16) (>1024) (16)
d
Kanamycin S S S S S S S S
(8-32)a (16) (16) (16) (32) (16) (16) (16) (8)
Gentamicind S S S S S S S S
(2-8)a (4) (4) (2) (8) (4) (4) (4) (4)
f f
Tetracycline I TRNG R R I TRNG I I
(0.25-32)a (0.25) (32) (2) (4) (1) (16) (1) (0.5)
Rifampicind S S S S R R S R
(0.125->32)
a
(0.125) (0.5) (0.5) (0.5) (>32) (>32) (0.25) (>32)
a
category of senstivity based on minimum inhibitory concentration (MIC, mg/l) using Etest. The range of the MICs for
each antimicrobial and the different strains are given in parenthesis. Most important, the precise MICs should be
interpreted with caution because these were derived using one specific method only and, accordingly, can slightly
differ using other methods. However, the identified resistance phenotypes (SIR-categorization) should be the same.
b
CMR, chromosomally-mediated resistance; PPNG, penicillinase-producing N.gonorrhoeae that should always,
independent of identified MIC-value, be considered resistant to penicillins.
c
NS, non susceptible, contains genetic resistance markers, but clinical/laboratory correlates are insufficient to
allow resistance phenotype designation.
d
Breakpoints from other sources than the Swedish Reference Group on Antibiotics (www.srga.com): ertapenem,
tentative based on wild type MIC-distribution; kanamycin; gentamicin; and rifampicin (www.bsac.org.uk).
e
LLR, low-level resistance; HLR, high-level resistance.
f
TRNG, tetracycline resistant N.gonorrhoeae (plasmid-mediated)
Reference: Unemo, M, Fasth O, Rfedlund H, Limnios A, Tapsall J, Phenotypic and genetic characterization of 2008 WHO
Neisseria gonorrheae reference strain panel intended for global quality assurance and quality control of gonococcal
antimicrobial resistance surveillance for public health purpose. J Antimicrob Chemother, 2009; 63;1142-51
TRNG 4
3.`WHO L 2 0mm Resistant 16 0mm 0mm Resistant 0.125 6mm Less <,=64 8mm Sensitive
sensitive
1 3
(CMRNG) HLR
4. WHO O 32 0mm Resistant 0.03 15mm 13mm Sensitive 0.016 12mm Sensitive >1024 0mm Resistant
(PPNG)2
5. WHO Q 1 0mm Resistant 1 2mm 0mm Resistant <,=0.03 8mm Sensitive <,=64 8mm Sensitive
TRNG 4 (CMRNG)1
1. CMRNG= Chromosomally mediated resistant Neisseria gonorrhoeae
2. PPNG = Penicillinase producing Neisseria gonorrhoeae
3. HLR = High Level Resistance
29
4. TRNG = Plasmid mediated resistance to Tetracycline (WHO G and WHO Q are TRNG strains)
Table. Australian Gonococcal Surveillance Programme Quality Control values for antimicrobial susceptibilities of
reference strains of Neisseria gonorrhoeae determined by MIC, agar plate dilution test method.
Penicillin 0.5 unit MIC2 0.008 0.5 >2.0 0.5 >2.0 2.0 2.0 >2.0 >2.0 >2.0 0.25 1.0
Category 3 S LS PPNG LS PPNG R R PPNG PPNG PPNG LS R
Ciprofloxacin 1 µg MIC 0.016 0.016 0.016 0.25 8 >16 16 2 4 0.03 0.016 1.0
Ceftriaxone 0.5 µg MIC <,=0.008 0.016 <,=0.008 <,=0.008 <,=0.008 0.06 0.125 <,=0.008 <,=0.008 <,=0.008 <,=0.008 <,=0.03
Category S S S S S LS LS S S S S S
Spectinomycin 100µg MIC 512 <,=64 <,=64 <,=64 <,=64 <,=64 <,=64 <,=64 <,=64 >1024 <,=64 <,=64
Category R S S S S S S S S R S S
Tetracycline 10 µg MIC <,=8 <,=8 <,=8 32 <,=8 <,=8 <,=8 <,=8 16 <,=8 <,=8 >32
Category Not TRNG Not TRNG Not TRNG TRNG Not TRNG Not TRNG Not TRNG Not TRNG TRNG Not TRNG Not TRNG TRNG
Azithromycin - MIC <,=0.5 <,=0.5 <,=0.5 <,=0.5 <,=0.5 <,=0.5 <,=0.5 <,=0.5 <,=0.5 <,=0.5 2 <,= 0.5
Category S S S S S S S S S S R S
Β-lactamase Negative Negative Positive Negative Positive Negative Negative Positive Positive Positive Negative Negative
S = Sensitive; LS = Less sensitive; R = Resistant; PPNG = Penicillinase producing N. gonorrhoeae; TRNG = Tetracycline resistant N. gonorrhoeae (High level tetracycline resistance, plasmid mediated).
Reference: Tapsall J and Members of the National Neisseria Network of Australia. Antimicrobial testing and applications in the pathogenic Neisseria. In: merlino J, ed., Antiimicrobial susceptibility testing:
methods and practices with an Australian perspective. Australian Society for Microbiology, Sydney, 2004. Pp 175-188.
30
1.10 Maintenance of bacterial strains
Reference strains of designated categories of susceptibility should generally be maintained by
the laboratory. The laboratory may also store certain clinical isolates of interest for reference.
The following storage methods may be used in the absence of facilities for storage by liquid
nitrogen.
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1.10.1 Preservation of bacteria by deep freezing (-70 C):
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Any of the following two methods can be used for preservation of Neisseria gonorrhoea at -70 C
for a long time.
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Sterilize by autoclaving at 15 psi (121 C) for 15 minutes. The medium used is distributed in
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0.7-1.0 ml aliquots in small test tubes and stored at 2-8 C prior to use.
b. Preservation Medium
Ingredients
1. Trypticase Soy Broth, Becton Dickinson 30 g
2. Yeast Extract 3.0 g
3. Agar No 2, Lab M 0.5 g
4. Distilled water 700 ml
5. Horse serum 300 ml
Method
1. Mix 1-4
2. Sterilse at 121O C for 15 mins
3. Cool Mixture to <50O C
4. Add horse serum and mix
5. Check pH and if needed, adjust pH to 7.5 + 0.1
6. Dispense in sterile tubes for freezing, volume 1ml
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7. Store at 2- 8 C
Preparation of bacterial suspension
Subculture on chocolate agar and make suspension from fresh 20-24 hr subculture. Use all the
material from a 90mm agar plate into 0.5-1ml of above medium.
Procedure: A fresh, pure culture of the gonococcus to be stored is heavily inoculated onto a 3 ml
volume chocolate agar slope in a polycarbonate (plastic) screw-top Bijou (5 ml volume) bottle
and incubated with the screw cap loosened for a minimum of 24 hours in a CO2 enriched
atmosphere or until visible growth is present on the agar surface. Sterile liquid paraffin is then
used to completely fill the agar slope, the screw cap lid is fully tightened and the Bijou bottle
slope is then stored at 35-36OC until the N.gonorrhoeae are tested or forwarded elsewhere.
When the gonococci are required for testing, a sterile bacteriological loop is inserted through
the paraffin overlay to remove the bacterial growth. When this is inoculated onto a fresh agar
plate and streaked, globules of paraffin will also be present. However after incubation, for 48
hours, gonococcal colonies are readily discernable and can be subcultured for appropriate
examination. The original paraffin overlaid slope can be returned to storage for further use, but
special care must be taken at the initial inoculation to use a pure culture and to ensure that
when sampled the slope is not contaminated.
Documenting location of strains: This should be recorded as should details of access to the slopes.
Note:
i) polycarbonate (plastic) bottles, not glass, must be used.
ii) larger volume containers may also be used e.g. 30 ml MacCartney bottles but these should
also be polycarbonate and will require larger volumes of bacteriological media and paraffin
and occupy more storage space.
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iii) the storage temperature should be 36 C, not room temperature, or else loss of viability will occur,
iv) for transport, lower temperatures will not affect viability for transit times of up to 5 days
v) loss of plasmids will occur on long term storage;
vi) it is suggested that familiarity with the system be obtained by both recipients and receivers
of the slopes before the system is implemented.
vii) fully viable gonococci have been recovered after nine months with this system, but there
are no data on viability beyond this period.
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viii) paraffin may be sterilized in large batches e.g. 200 ml placed in a hot air oven at 180 C for 2 hours.
ix) it is important that the slopes be completely filled with paraffin and that there are no air bubbles.
1.10.3 Lyophlilization:
Prepare 7.5% glucose horse serum (7.5 gm of glucose to 100 ml horse serum) sterlize and put 1
ml in each test tube. Inoculate each tube with a very heavy growth from half a plate, vortex,
distribute into 3 ampoules, freeze and lyophilize.
SUMMARY
1. Direct microscopic examination of Gram stained slides is done at the peripheral level. More
than 95% of the males with symptoms can be diagnosed by grams staining alone. Grams
stain is not a recommended method of diagnosis in female patients.
2. Specimens from women and men should be cultured, preferably on two media, one with
inhibitors and one without it. For all cultures, colony morphology, oxidase reaction, superoxol
test and Gram negative diplococci in smear made from culture and rapid carbohydrate
utilization test (RCUT) confirm the diagnosis. β-Lactamase test can also be done.
3. Antibiotic sensitivity testing should be done at central level regularly.
Chlamydia trachomatis is an intracellular organism. It causes lower genital tract infection, such
as non-gonococcal urethritis and cervicitis, and may cause PID and endometritis (serovar D to
K). It can cause inclusion conjunctivitis. Three specific serovars (L1 to L3) cause
lymphogranuloma venereum (LGV), while other serovars (A to C) cause trachoma. Smear and
staining do not help in the diagnosis. Tissue culture (gold standard), antigen detection (DFA and
ELISA), nucleic acid hybridization and amplification methods are used for laboratory diagnosis.
The current reference test (expanded gold standard) for diagnosing C.trachomatis infection is
commonly consistent result with two non-culture techniques. The culture procedure, however,
is expensive, slow, labour-intensive, technically difficult and beyond the capacity of most
laboratories. Culture is not suitable for widespread screening and is mostly carried out in
research laboratories.In competent hands, the specificity of culture is 100 percent, its sensitivity
is estimated to be no more than 70 to 85 percent compared to DNA amplification. Cervical
culture for C.trachomatis has a 65-90 percent sensitivity compared to DNA amplification of first-
catch urine. Other non-culture techniques (DFA, EIA, DNA hybridisation) developed during the
1980s, which are easier to perform and less expensive than culture, are still widely used.
Bubo pus - viscous material should be ground and suspended in nutrient broth or cell culture
medium. This should be tested for bacterial contamination and then inoculated into cell
cultures for isolation of Chlamydia.
Rectal swabs - As they are likely to be contaminated with gut flora, suspend the swabs in cell
culture medium containing gentamycin, vancomycin. Shake the suspension to mix, centrifuge
for 10 mins at 300g. Inoculate supernatant onto cell culture medium.
Tissue samples for culture - to be transported in dry ice in a frozen condition to central
laboratory. Thaw, mince with sterile scissors, grind with a homogeniser. Add cell culture medium
to make a 20% suspension and incubate.
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Transport swabs at 4 C in ice within 4 hours of collection. If it cannot be cultured within 48 hours,
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freeze at -70 C.
Mc Coy, Hep2 or Hela cells are commonly used for culture of Chlamydia. Before inoculating
samples, pretreat the cell monolayers with 30µg/ml of Diethylaminiethyl-Dextran in Hank’s
balance salt solution for 20 mins. Clinical specimens are shaken with sterile 5 mm glass beads to
lyse the epithelial cells and release the chlamydiae before being used for inoculation. For
inoculation, the medium is removed from the cell monolayer and 0.1 mL to 1 mL of inoculum is
added to the cells. The specimen is centrifuged onto the cell monolayer at approximately 3000 g
at room temperature for 1 h.
Shell vials are incubated at 35OC in 5% CO2 for 2 h to allow for the uptake of chlamydiae. The
medium is then discarded and replaced with medium containing 1 µg of cycloheximide/mL. The
cells are incubated at 35OC in 5% CO2 for 48 h to 72 h, and one cover slip is examined for
inclusions by immunofluorescence, iodine staining or Giemsa staining. Immunofluorescence is
the preferred method because it is more specific than Giemsa staining and can give a positive
result as early as 24 h post inoculation.
Giemsa staining:
Giemsa staining has very poor sensitivity.
Reading:
Chlamydial elementary bodies - Bright apple-green pinpoints.
Cells - Red
Interpretation:
Positive: > 10 Elementary Bodies / slide
Doubtful positive: < 10 Elementary Bodies / slide
Negative: No Elementary Bodies
Actual instructions for the test procedure are supplied with the kit to be used and should be
followed as such. This is the only test available for simultaneous assessment of specimen
adequacy by visualization of cuboidal columnar epithelial cells and is sensitive and specific in
symptomatic patients.
Internationally approved NAATs are recommended for use and in-house tests should be
validated against internationally approved NAATs. The facilities for these should be established
in regional reference laboratories for diagnostic purposes because of their higher sensitivity and
specificity. Different type of genes such as MOMP gene, Plasmid DNA, r-RNA - 16S & 23S and
phospholipase gene are targeted for diagnosis by NAATs. Some of the NAATs targeting the
common endogenous plasmid can give false negative results with a new variant of
C.trachomatis (nvCT) strain which has been recently isolated in Sweden (2006) and this variant
has a 277 bps deletion in a portion of the plasmid. Therefore, the choice of sequence is crucial.
Multiplex PCRs to detect C.trachomatis and N.gonorrheae are useful for detection of both these
organisms with good sensitivity and specificity.
Syphilis is caused by the bacterium, Treponema pallidum, which is a Spirochaete. It occurs due
to the contact of an infectious lesion with the mucous membrane or injured skin. Culture of
T.pallidum is not feasible. So the clinical diagnosis of syphilis is confirmed by i) seeing the
organism in the exudate from ulcers; or ii) by finding antibodies to this pathogen in serum or
cerebrospinal fluid.
Classical syphilis occurs in stages- Primary, Secondary and Tertiary stages, with a latent stage in
some cases . In the early stage of the disease (primary syphilis) T. pallidum can be seen in ulcers
in the genital or rectal area . Antibody can be detected from 1 to 4 weeks after the formation of
the ulcer, therefore, seeing the organism in the lesion provides the most reliable method of
diagnosis in the early stage of the infection.
Test Principles
Clinical diagnosis of primary syphilis is confirmed by using dark field microscopy to demonstrate
Treponema pallidum in material from suspected lesions. A positive result is an almost certain
diagnosis of syphilis.
Specimen Collection
Materials required:
1. Dark field microscope
2. Thin glass slides (1mm)
3. Thin cover slips (.1mm)
4. Glass marking pencil / marker
5. Normal Saline
Dark field examination of oral lesions is not recommended. All positive dark field tests with
mouth specimens must be confirmed by a direct fluorescent antibody test. The indigenous flora
of the oral cavity frequently contain a spiral organism, T. denticola, which is indistinguishable
from T. pallidum. It can give false-positive results.
3. Cervical/vaginal lesions
a. With visualization by a bivalve speculum, remove any cervical or vaginal discharge.
b. Obtain serous exudate with a sterile bacteriological loop.
c. Prepare slides as described above.
5. Lymph node
a. Disinfect the skin over the node by swabbing it with iodine and alcohol or another
suitable agent.
b. Rinse a sterile 20 gauge needle and a 2ml syringe with sterile physiological saline.
c. Allow 0.2 ml or less of the saline to remain in the syringe.
d. Hold the node firmly and insert the needle well into the node. The ability to
manipulate the node freely with the needle tip is a good indication that the capsule of
the node has been pierced.
e. Inject the sterile physiological saline into the node.
f. Macerate the tissue by gently manipulating the needle in various directions.
g. Aspirate as much fluid as possible.
h. Discharge the aspirated material onto slides for immediate examination.
However, this test does not differentiate between T. pallidum and other pathogenic treponemes
causing yaws, endemic syphilis and pinta.
For detailed steps, the laboratory is advised to follow manufacturer’s instructions. As the test
requires fluorescent microscope, this test would not be available at primary level. This may be
done at intermediate level laboratories.
Non-treponemal tests:
RPR and VDRL are the two most commonly used non-treponemal tests.
Test method:
1) Take one test card. With the pipette place 0.05 ml of unheated serum on one of the circles.
2) Spread the sample on the circle with the disposable plastic stirrer.
3) Test known positive and negative sera along with the unknown samples.
4) Gently shake the RPR card test antigen and put one drop antigen in the serum sample on the circle.
5) Rotate the card on the mechanical rotator after adjusting the speed. The duration of the
rotation should be as per the instruction with the kit.
6) Remove the card from the rotator and observe the result immediately with naked eye in
bright light.
7) Reading:
Small to large clumps - Reactive
No clumps or slight roughness - Non- Reactive
If clumps occur, perform the tests in serial dilutions starting from 1in2,4,8,16 and so on. Find out
the last dilution in which clumping occurs.
Reagents:
1) VDRL antigen: supplied by Institute of Serology 3, KYD Street , Kolkata should be used.
2) VDRL buffered saline: supplied by the manufacturer. It has 1% sodium chloride, pH 6.0 + /-
0.1 (Formaldehyde 0.5 ml, Na2HPO4 3.037 gm, KH2PO4 0.170 gm, NaCl 10.0 gm, distilled
water 1000 ml).
3) Normal saline (0.9%)
Preparation of Serum:
1) In a serological water bath with a temperature of 56O C, heat the specimen serum for 30 minutes.
2) After heating, examine the sera and again centrifuge any serum sample which shows particles.
3) If the test is not done for 4 hours after heating the serum, again heat the serum sample at
56O C for 10 minutes before carrying out the test.
4) Perform the test when the sera cool down to room temperature.
Procedure:
1) In summer, carry out the test early in the morning and in winter, in the afternoon so that
the test is done at the prescribed room temperature of 23 - 29OC.
Reading:
Medium and Large clumps - Reactive
Small clumps - Weakly Reactive (WR)
No clumping or slight roughness - Non-Reactive
Procedure:
1) Take 6 test tubes and keep them in a rack.
2) Pipette 0.5 ml normal saline in each tube.
3) Pipette 0.5 ml of test serum in tube 1 and mix well. (Serum dilution = 1:2).
4) Take 0.5 ml of diluted serum and add to tube 2. Mix well and transfer 0.5 ml to next tube. In
this way mix well and go on adding 0.5 ml of diluted serum to each next tube till tube 6 is
reached. Mix well and discard 0.5 ml of diluted serum from the last tube. The dilution
obtained in the 6 tubes are 1:2, 1:4, 1:8, 1:16, 1:32, 1:64.
5) Take a VDRL slide and add 0.5 ml of serum from the sixth tube on ring 7.
6) Similarly add 0.05 ml of serum from the tube no 5, 4, 3, 2 and 1 in the wells 6, 5, 4, 3 and 2
respectively.
Reporting:
Procedure:
1. Pipette 0.5 ml normal saline in each well of the slide starting from well 2.
2. In well 1, take 0.05 ml of neat, undiluted serum as for the qualitative test.
3. Pipette 0.5 ml of test serum in well 2 and mix well. (Serum dilution = 1:2).
4. Take 0.5 ml of diluted serum from well 2 and add to well 3. Mix well and transfer 0.5 ml to
Reading:
Visible clumping - Reactive (R)
No clumping or very slight roughness - Non-reactive (NR)
Biological False Positive (BFP) reactions are defined as positive reaction in a cardiolipin test and
a negative reaction in a confirmatory treponemal test. Quantitative RPR/VDRL test with a titre
≥ 1:8 is considered as significant titre and is suggestive of syphilis. It is recommended that a
confirmatory test such as TPHA should be performed on all sera with a reactive RPR/VDRL
regardless of its titre especially to accurately diagnose and confirm syphilis in case having titres
<1:8. Some of the TPHA positive cases with RPR/VDRL titre <1:8 may be either because of
biological false positivity or representative of early / latent / late syphilis cases or treated cases
of syphilis. In addition, combination of this testing strategy (RPR/VDRL + TPHA) is less expensive
and much easier to perform. BFP reactions can be observed in various acute and chronic
conditions in the absence of syphilis. These can be acute, giving positive reaction for few weeks
to few months or chronic positive RPR/VDRL reaction lasting more than six months. Examples of
BFP reactions are; infections, injuries, inflammation and early HIV infection, SLE, collagen
vascular diseases, leprosy ,malaria relapsing fever, hepatitis, Infectious mononucleosis, tropical
eosinophilia etc.
In this test, red blood cells coated with T.pallidum are mixed with the serum of patients. If
antibodies to T.pallidum are present the red cells will agglutinate in the form of a smooth matt at
the bottom of the microtitre plate well. If no antibodies are present then no agglutination
occurs and the red cells settle at the bottom of the well in button form.
Many TPHA kits are available commercially. Test procedures are different for each kit. For
detailed information see the instructions provided with the kit.
Incubate
Agglutination No agglutination
(antibody in serum of patients with syphilis): (no antibody):
Irregular deposition of RBCs in the form of a Clear button type deposit of RBCs.
smooth matt, overing the well.
Incubate.
Fluorescein conjugated anti human globulin serum
Dark ground condenser with UV microscope / Fluorescent Microscope
According to some studies when results are reactive to the treponemal test but non-reactive to the
RPR test, persons with history of previous treatment will require no further management. For person
without the history of treatment, a second, different treponemal test should be performed. If the
second treponemal test is non-reactive, the clinician may decide that no further evaluation or
treatment is indicated, or may choose to perform a third treponemal test to resolve the discrepancy.
Several rapid tests for syphilis are now commercially available. These are simple point of care
tests that can be performed outside a laboratory setting with minimal training and no
Thus, given the anticipated increase in use of treponemal tests for screening, the introduction of rapid
tests in a country or a specific area, should only be under taken after due consideration to the following
aspects- Test performance, ease of use, format of the testing, shelf life and temperature stability,
requirement of additional supplies for testing, access to the test, quality of testing, availability of
treatment to sero reactive cases and cost. Further, the epidemiology of disease in various countries,
the different patterns of sero reactivity and systems of health care have to be ascertained.
Hence, countries that have already established effective syphilis control programmes, including
screening for antenatal and high risk populations, should preferably maintain their programme
rather that introduce rapid tests.
SUMMARY
1. Immediate diagnosis of T.pallidum infection by dark field microscopy makes it a very
useful diagnostic tool. So this test should be done in all laboratories at the intermediate
and tertiary centres.
2. A simple and quick serological test like rapid plasma reagin (RPR) card test should be done
at the peripheral level.
3. VDRL test is the test to be done at the intermediate and central levels. Quantitative VDRL
test should be done to find the actual titre of the antibody. VDRL on CSF must be done to
diagnose neurosyphilis.
4. In a central laboratory a confirmatory test like TPHA should be done. With due consideration
to cost constraints, if available, test may be done at the intermediate level also.
5. The Fluorescent treponemal antibody absorption (FTA-Abs) test is very specific and helps
in early diagnosis of syphilis. It is better than haem-agglutination assay. Experienced and
highly trained staff are needed to do the test, which is also time consuming. Because the
test is costly, it is done only to confirm the result of other tests at the central laboratory.
4.2. Tests that can be done to diagnose infection due to H.ducreyi are:
Sample Test
Swab from ulcer, Aspirate from ulcer/bubo Gram staining and microscopy
Swab from ulcer, Aspirate from ulcer/bubo Culture, β-lactamase testing and antibiotic
sensitivity
Method:
1. Ulcer: Clean the ulcer with sterile swab moistened with saline to remove the dry crust.
2. Collect the exudates from the ulcer base with a sterile swab moistened with saline by
rubbing vigorously at the base of the lesion.
3. Abscess: Disinfect skin with alcohol and iodine. Aspirate fluid with a needle and syringe.
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4. Prepare smear on a clean, grease free microscope slide by rolling the swab stick 180 in
one direction. This preserves the arrangement of the organisms.
5. Stain by Gram's method.
6. Observe under oil immersion lens of light microscope.
Method:
1. It is advisable to inoculate two different selective agar plates at the point of sample
collection itself.
2. Inoculate the material on both isolation media and send to the laboratory immediately for
incubation.
3. If sample is transported from a primary level laboratory, transport swabs/aspirates in
Amies transport medium (Annexure-II) . Transport at 4OC and make sure that the sample
reaches the culture laboratory within three days.
4. Keep the inoculated media in a candle jar with moist chamber and incubate at 32 -34OC.
5. Read the cultures after 48 hours incubation.
6. If negative after 48 hours, keep up to 5 days before finally calling it negative.
Reading of cultures:
Small colonies up to 0.5 mm in diameter.
Greyish yellow, granular, raised non-mucoid colonies, translucent to opaque.
Typically, the colonies move intact on the surface of the media when pushed with a platinum
loop.
Difficult to pick up the colony from the media.
5.4. Procedure
Clean an area of granulomatous tissue in the red lesion preferably near the edge with a gauze
soaked in saline and finally with a dry gauze.
5.4.2 Prepared smears can be examined microscopically under oil immersion lens of a light
microscope after staining.
} Giemsa, Leishman or Wright's stain are all satisfactory.
} For rapid staining, Rapi Diff stain can be used.
} For fixed or embedded tissue sections, Giemsa or silver stains are preferred,
haematoloxylin and eosin is not satisfactory
RapiDiff Stain
} Place the smear in RapiDiff fixative for 15 seconds (6 dips)
} Then place it in solution RapiDiff 1 (Eosin Y) for 15 seconds (6 dips)
} Place in solution RapiDiff 2 (Thiazine Dye Mixture) for 15 seconds (6 dips)
} Finally rinse the slide in phosphate buffer pH 6 8.
} Air dry the stained slides and examine by light microscopy under oil immersion (1000 x
magnification). (Annexure-1)
Others tests
} Polymerase chain reaction techniques may
be more sensitive; however, they are
currently only used for scientific research.
} An indirect immunofluorescent technique is
available to test serum; however, it is not
accurate enough for confirmatory
diagnosis.
} Papanicolaou smears may identify Donovan bodies in patients undergoing routine
cervical cytological screening.
} If bony involvement is suspected in granuloma inguinale, radiography or other
imaging studies are indicated
} Testing for other sexually transmitted diseases is warranted because multiple
coexisting infections are common.
SUMMARY
1. Direct microscopy is performed by impression smears or crushed tissue between two
slides and staining by Giemsa stain.
2. K. granulomatis does not grow on any standard microbiological media in the lab.
The altered vaginal flora is characterized by the presence of the following bacteria:
} Gardnerella vaginalis
} Mobiluncus mulieris
} Mobiluncus curtissii
} Bacteroides sp.
} Peptostreptococcus sp.
} Prevotella sp.
The patient comes with the symptoms of foul smelling (due to the release of amines produced
by anaerobic bacteria that decarboxylase amino acids like Iysine and arginine), fishy vaginal
odour. Grey homogeneous, thin vaginal secretion at the vaginal opening is usually seen.
Method:
1. Collect the specimen with gloved hands and vaginal speculum in place.
2. Collect the specimen from the lateral or posterior fornix of vagina with a sterile cotton
wool swab soaked in normal saline.
6.2. Procedures
A. Vaginal pH test:
1. Take pH indicator paper strips with a range of ± 3.8 to ± 6.0.
2. Touch the specimen swab on the pH paper, or
3. Touch the pH paper to the tip of the vaginal speculum after removing it
from the vagina or touch the pH paper to the wall of vagina directly.
4. Do not allow the contact of pH paper with cervical secretions.
Reading:
} Normal adult vagina has an acid pH of 3.6 to 4.2
} In Bacterial vaginosis the pH is raised to 4.5 or more.
NB.Presence of menstrual blood, cervical mucus, semen or T. vaginalis infection may also
raise the vaginal pH.
C. Microscopy
1. Wet mount examination:
a. Take a drop of vaginal fluid on a clean grease free microscope slide and mix with a
drop of normal saline. Alternatively take a drop of normal saline on a clean grease
free microscope slide and gently rub the swab over it to mix the vaginal fluid with it.
b. Put a coverslip on the mixture and see to it that the preparation is free of air
bubbles and uniformly spread.
c. Observe the slide under 40 x magnification of the objective.
Reading:
Presence of “Clue cells” (> 20%) suggests the diagnosis.
A clue cell is a squamous epithelial cell with many coccobacillary organisms attached to its
surface giving it a granular appearance. The cells do not have a well defined edge because of the
presence of the bacteria and disintegration of the cell. Over 20 % clue cells are seen along with
the normal vaginal epithelial cells in a case of bacterial vaginosis. In most of the cases the
organism attached to the epithelial cells is G. vaginalis. Figure 5 -
Wet mount showing clue cells
2. Gram Stained Smear:
a. Prepare a smear on clean grease free microscope
slide by rolling the swab on the slide.
b. StainthesmearwithGramstainafterdryingandheat fixing.
c. Observe the smear under oil immersion lens after
placing a drop of liquid paraffin on the slide.
At least 5 oil immersion field (OIF) should be examined to determine an average of each
morphotype. Total score = Lactobacilli + G.vaginalis and Bacteroides spp. + Gram variable rods.
30 or> 0 0 0 0 0
5-30 1 <1 1 <1 1
1-4 2 1-4 2 1-4 1
<1 3 5-30 3 5-30 2
0 4 30 or > 4 30 or> 2
Interpretation:
score Report
To conclude, a stained smear gives a better idea about the normal vaginal organisms and the
identification of ‘Clue cells’ is better by Gram-stained method as compared to wet preparation.
SUMMARY
1. Wet mount examination and the pH test can be done at the peripheral level for bacterial
vaginosis.
2. The pH test, KOH test, wet mount examination and Gram staining can all be done at the
intermediate and central level.
3. Using the Nugent’s criteria, the sensitivity of Gram stain for diagnosis of BV is very high.
Since Candida is part of normal flora, serological tests or nucleic acid hybridisation or
amplification tests do not play a diagnostic role as they can lead to false-positive results.
In Females:
1. Collect scrapings from vagina and edges of erythematous (red) lesions of the vulva,
with sterile cotton wool swabs soaked in saline.
2. Collect pooled vaginal discharge from the posterior fornix in a sterile container.
3. Collect urethral swab if necessary.
In Males:
Swab from the sub – preputial (area under prepuce) area and urethra is collected, if necessary.
Gram Staining:
1. Prepare a smear on a clean, grease free
microscope slide.
2. Heat fix the slide (Annexure-I).
3. Stain the slide by Gram’s method (Annexure-I).
4. Observe under oil immersion lens of light microscope.
Reading:
Gram positive (violet) budding yeast cells (looking like figure of 8) and yeast hyphae.
Reading:
Observation of germ tube confirms the presence of C. albicans. A germ tube is a short,
lateral hyphal extension (filament) of the yeast cell and is not constricted at the base.
Reading:
Hyphae and pseudohyphae with clusters of blastospores (4-5µm) at internodes. Terminal
chlamydospores, 8-12µm. Formation of Chlamydospores is characteristic of C. albicans.
Occasionally C. stellatoidea may also form chlamydospores. Further confirmation is then
done by carbohydrate assimilation test.
Reading:
A growth ring of the yeast and a change in indicator colour from purple to yellow –
positive test. Carbohydrate is assimilated.
No growth and no change in the indicator colour – Negative test. The isolate lacks
enzymes to assimilate that particular carbohydrate.
For carbohydrate assimilation reactions of C. albicans and C. stellatoidea refer table below:
Organism
Carbohydrate
Candida albicans Candida stellatoidea
Dextrose + +
Maltose + +
Sucrose + -
Lactose - -
Galactose + +
Trehalose + +
Raffinose - -
Cellobiose - -
Melibiose - -
Inositol - -
Xylose + +
C. dubliniensis Green --
C. firmetaria Pink to lavender, pale border Some flat, rough; others waxy
SUMMARY
1. Potassium hydroxide wet mount examination should be done at the peripheral level for
suspected Candida spp. infection.
2. Gram stain may also be performed at the peripheral level for suspected Candida
infections.
3. Confirmation of Candida infection by culture can be done at the intermediate and central
level laboratories. Culture should only be done when candidiasis is clinically suspected
while the wet mount is negative.
4. Speciation of Candida can be done at the intermediate or central level. This requires
culture of the organism.
5. There is no indication for routine susceptibility testing for candida. It may be done at the
reference laboratories to review treatment recommendations from time to time
Hence combination of various microbiological tests should be employed for its diagnosis.
Suitable clinical specimens for laboratory diagnosis:
In females:
} High vaginal swabs from posterior fornix and lateral walls
} Vaginal secretions
} Endocervical smear/secretions
In males:
} Centrifuged deposit of urine
} Urethral discharge
In females:
a. With the help of a vaginal speculum visualize the posterior fornix.
b. Collect the discharge from the posterior fornix with a sterile cotton wool swab soaked
in normal saline.
In Males:
Provide the patient with a sterile bottle and ask him to collect first 20 ml of the urine
passed first time in the day (early morning).
8.4 Microscopy
A. Wet Mount:
The wet mount should be examined within 15-20 minutes of collection as the parasite
loses its distinctive motility due to variation in temperature.
Procedure:
} Suspend the secretions in 0.85% normal sterile saline
} Vigorously rotate swab in saline
} Press against the side of the tube to express as much fluid as possible
} Place one drop of the expressed fluid on a clean glass slide with a cover slip
} Examine the wet mount under the microscope
From urine prepare the wet mount by first centrifuging the urine at 1500 RPM for 10 minutes
and then taking a drop of the deposit on a clean grease free microscope slide.
Observe the slide first under 10 x magnification. Any field which shows the suspected
organism is then seen under 40 x magnification of light microscope.
Centrifuged samples do not show motility because the flagella are detached during
centrifugation.
Specificity: 100%
Sensitivity: 35-80%.
4
Minimum concentration of 10 organisms per ml. of vaginal fluid appears to be necessary for
identification of the protozoan by wet mount.
Wet mount examination may be employed routinely despite limited sensitivity because
} It is inexpensive
} It is quick
} Patients can be treated during the first visit
B) Staining:
} Prepare a smear of infected material on a clean grease free microscope slide.
} Allow it to dry in air, fix with methanol and stain.
} Giemsa, Papanicolaou, periodic acid-Schiff, acridine orange, fluorescein and
immunoperoxidase stained smears can be used to detect T.vaginalis in
asymptomatic women during routine examinations.
} Detection using the Papanicolaou test has reported sensitivity of 60% and
specificity of 95–97%. Papanicolaou (Pap)(Annexure-I) staining may be the most
practical approach for the detection of asymptomatic infections as a large number
of asymptomatic women undergo routine cytological screening.
} Observe under oil immersion objective of light microscope after placing a drop of
liquid paraffin on the smear.
} This method is less reliable than the wet mount for diagnosis.
8.5 Culture
a. Culture is gold standard for the diagnosis of trichomoniasis.
b. Perform culture in cases where vaginal wet mount preparation is negative and for
Interpretation
At intervals, macroscopically look for turbidity and microscopically examine the wet mount
prepared from the material taken from the bottom of the tube for motile trichomonas. Giemsa
stain may be performed to see the characteristic morphology.
The medium is equally suitable for the examination of urethral and vaginal swabs, and urine
specimens.
Advantages:
} Simple procedure.
} Urine can be used instead of urethral specimen.
} Can be stored at room temperature up to one year .
} More sensitive than culture on other liquid/semisolid/solid media.
Procedure:
1. Collect the specimen from the posterior fornix of the vagina with a sterile swab.
2. Inoculate it in the top pouch of In Pouch for culture of T. vaginalis (InPouch TV is a
double pouched container made of soft plastic).
3. The specimen gets introduced into the bottom pouch immediately after collection.
4. Conduct microscopic examination of the bottom pouch when the specimen arrives in
the laboratory (no examination is made of the top pouch because its contents had
been immediately pushed into the bottom pouch).
5. Incubate the cultures at 35OC.
6. Examine for motile T. vaginalis at 24, 48 and 96 hours of incubation by using a 10X
objective directly through the pouch.
7. Internal quality control for T. vaginalis InPouch culture is made by incubating one
InPouch per batch as a sterility check on reagents and inoculating one pouch with a
known culture of T. vaginalis to check the quality of the batch of InPouch.
Top
Pouch
Bottom
Pouch
Rapid tests based on color immunochromatographic, capillary flow, "dipstick" technology are
available, that detect antigens directly from vaginal swabs. The test is performed by inserting a
vaginal swab into a test tube with special buffer, mixing the solution vigorously by hand,
removing the swab, and then inserting a test strip. A visible blue test line is detected if
Trichomonas antigen is present. No microscope is needed. Results are available within 10
minutes of administration.
SUMMARY
1. Observation of the parasite in the saline wet mount of the vaginal or urethral secretions
confirms the diagnosis and can be done at the peripheral and intermediate level.
2. Microscopy confirms diagnosis in over 60% of symptomatic women.
3. In asymptomatic women and men, culture in liquid TV culture system, can be done at the
intermediate and central level, especially in suspected patients. Culture can also be done
in patients with clinically suspected infection, but with a negative wet mount
examination.
For microscopy:
1. Excise the top of a vesicle with a fine sterile scalpel or scissors.
2. Scrape the bottom of the ulcer with a scalpel or curette .
3. Prepare a smear on a clean, grease free microscope slide and allow it to dry in air.
4. Fix the smear with methanol.
For culture:
1. Open a large vesicle with a small (26) gauge needle.
2. Aspirate the fluid with a tuberculin syringe.
3. In women collect specimen from vagina with a sterile cotton wool swab.
4. Transfer the material into a vial containing 1ml of transport medium (Annexure-II)
5. Store at 4OC if the culture is to be performed within 48 hours.
6. Freeze at -70OC if the culture can only be done after 48 hours after collection.
Immunoflouresent staining:
1. Method of choice for direct diagnosis of viral infection.
2. Kits are available for this technique. So only a main outline of the procedure is given.
Reading:
Many yellow or yellow green shining (fluorescing) cells are observed.
Negative IF Positive IF
These methods have largely been superceded by more sensitive and less laborious procedures
which utilize amplification of the target HSV DNA by polymerase chain reaction (PCR).
Specificity of the amplification method is assured by either undertaking a second PCR with
target-specific primers (nested PCR) or by HSV-specific probe hybridization of amplified
products. In the case of possible genital herpes, PCR detects viral DNA for several days after
lesions do not contain demonstrable infectious virus . This may mean that a laboratory switching
to sensitive procedures based on nucleic acid amplification may have an increased number of
positive results on lesion samples with possible clinical dilemmas regarding the relevance of
positive results obtained after treatment.
With the recent advances in automation and kit developments for HSV detection and typing by
PCR (eg, Real Art HSV1/2 kit from Artus-Biotech USA), it is likely that this methodology will
become more widely used for routine diagnostic purposes.
Human papilloma viruses cause genital warts (Condylomata acuminate). More than 100 types
of HPV exist, more than 40 of which can infect the genital area. Most HPV infections are
asymptomatic, unrecognized, or subclinical. Oncogenic or high-risk HPV types (e.g., HPV types
16 and 18), are the cause of cervical cancers. These HPV types are also associated with other
anogenital cancers in men and women, including penile, vulvar, vaginal, and anal cancer, as well
a subset of oropharyngeal cancers. Nononcogenic, or low-risk HPV types (e.g., HPV types 6 and
11), are the cause of genital warts and recurrent respiratory papillomatosis. Asymptomatic
genital HPV infection is common and usually self-limited; it is estimated that more than 50% of
sexually active persons become infected at least once in their lifetime.
Diagnosis of genital warts is usually clinical, made by visual inspection. Genital warts can be
confirmed by biopsy, which might be indicated if 1) the diagnosis is uncertain; 2) the lesions do
not respond to standard therapy; 3) the disease worsens during therapy; 4) the lesion is atypical;
5) the patient has compromised immunity; or 6) the warts are pigmented, indurated, fixed,
bleeding, or ulcerated. Genital warts are usually asymptomatic, but depending on the size and
anatomic location, they might be painful or pruritic.
10.3 HPV does not grow in cell culture. Hence culture is not available.
10.4 Detection of HPV DNA by nucleic acid hybridisation and amplification. This can be made
available in central laboratory. Currently molecular methods of detection and genotyping of
HPV has become the gold standard. There are three types of assays.
1. non-amplified hybridization assays such as Southern transfer blot, Dot-blot
hybridization and in-situ hybridization.
Many commercial assays are available based on the following principle for detection of HPV
infection.
Hepatitis B virus is a complex DNA virus having three different antigens. These are Hepatitis B
surface antigen (HBsAg), Core antigen (HBcAg) and ‘e’ antigen (HBeAg). Presence of HBsAg in
saliva, seminal fluid and vaginal secretions indicates its potential for spread during sexual
activity.
Though hepatitis B infection covers many medical specialties, this manual is confined to its
testing as part of STIs only.
Diagnostic testing
At primary level, immuno-chromatography or any other point of care test (follow procedures as
per manufacturer technical instructions in the kit) can be used as a screening test for HBsAg. At
intermediate laboratory level, ELISAs, or Chemiluminiscence assays could be employed.
Recently Enzyme Immuno Assay for quantitation of HBsAg has been approved and is expected
to be performed at Intermediate level laboratories, which do not have molecular testing
facilities. For further confirmation on activity of infection and detection of viral nucleic acid,
refer samples/patient to central laboratory where the test is done.
Hepatitis C is a liver disease caused by the hepatitis C virus, which is a RNA virus. Though not
primarily a sexually transmitted infection, it is also reported to be transmitted through sexual
contact, exposure to infectious blood, blood products and organ transplants, injections given
with contaminated syringes and needle-stick injuries in health-care settings, IV drug use, being
born to a hepatitis C-infected mother. The virus has remarkable capacity to remain chronic and
also be responsible for development of hepatocellular carcinoma.
Nucleic acid testing- Real Time -PCR or TMA Serum/ EDTA Blood
detection (Transcription Mediated
Amplification)
Laboratory workers are at high risk for exposure to infectious agents. Infections can be acquired
from exposure to contaminated blood, tissue and other samples during routine processing.
Laboratory practices that reduce the risk of infection are standard precautions, specific good
laboratory practices, use of PPE, safety devices, proper decontamination, segregation and
disposal of waste materials.
1000 20 980
5000 100 4900
1. It is essential that all the glassware used for testing are cleaned well and are free of
contaminating organisms, dust and extraneous interfering substances. Most of the containers
(Petri dishes, tissue culture bottles, flasks, beakers etc.) and measuring devices (pipettes,
measuring cylinders) used in a standard laboratory are of glass. So proper washing and
sterilization of glassware is necessary to maintain high standards in an STI laboratory. If the
glassware is not cleaned and sterilized properly then the results obtained after using this
glassware will be erroneous. The glassware when new will be contaminated with spores and
when used glassware is again utilized it may be contaminated with traces of previous culture.
Hence the need for thorough cleaning and sterilization of glassware.
B) Old glassware:
1. Clean the glassware with soap solution.
2. Wash 6 times with tap water.
3. Place the glassware for 1 hour in a large plastic bucket in distilled water with 0.05%
IN HCI ( This neutralizes the alkalinity resulting from the use of soap)
4. Wash 3 times with distilled water.
5. Dry the glassware, wrap in aluminum foil and sterilize by dry heat at 160OC for 1
hour.
Maintenance of equipment in good working condition is absolutely essential for the smooth
functioning of laboratory. Preventive maintenance and calibration of equipment are generally
done by authorized service personnel. Routine maintenance and function checks are done by
the laboratory technicians who work with the equipment, under the supervision of technical
officer or laboratory in-charge.
All the equipment should have adequate documentation on its maintenance, repairs, downtime
and calibration. Equipment history card should be maintained for every equipment and it
should have:
} Name of the equipment
} Unique ID , model and the serial no
} Manufacturer name and address
} Service person’s name, address and contact no
} Date of installation
} Calibration frequency, calibration certificates
} Repairs and service details
} Every equipment should have work instructions for its operation and troubleshooting
guide available at the workplace.
Calibration of equipment:
All the equipment except centrifuges and micropipettes should be calibrated annually. The
laboratory should have an annual calibration plan which helps in timely calibration of
equipment. Centrifuges and micropipettes should be calibrated every six months . All
calibrations should be done by the manufacturer or an accredited calibrating
agency/laboratory. Records should be maintained.
Some of the common equipment and their maintenance protocol is given below:
1. Microscopes (Light, Dark field and Fluorescent): Every time the microscope is used, the
lenses should be cleaned with a lens paper/soft gauze soaked in xylene. Alcohol should
not be used to clean the optical system because it dissolves the cement material and
thereby detaches the lenses. The whole microscope can be cleaned with a clean gauze to
get rid of the dust particles, every time it is used. Special care should be taken to avoid
damage to the light source of dark field and fluorescent microscope. When the
microscope is not in use, use of a dust cover is desirable. Optical system should be cleaned
by a trained/ authorized person atleast once in six months AMC / CMC is desirable.
2. Centrifuge: It should always be placed in a horizontal position, the safety cover should be
closed when in use. It should be cleaned regularly with gauze. Calibration of the centrifuge
should be done every six months for the speed and timer with a calibrated tachometer.
1. Quality Assurance
Quality assurance indicates an overall plan of activities that are undertaken to demonstrate that
quality is ensured in the entire process flow so that the results are accurate and reproducible.
This plan consists of:
1. Organization and management
2. Availability of validated standard operating procedures
3. Document control, proper recording of data
4. Training and competency assessment of staff
5. Equipment maintenance and calibration
6. Monitoring of purchase and inventory
7. Environment and personal safety
8. Internal quality control procedures
9. Participation in external quality assurance programs.
10. Procedures to identify non-conformities, complaints and take corrective action
11. Risk assessments and preventive actions
12. Quality improvement through indicators
The QA program ensures that correct procedures are followed throughout pre-analytical,
analytical and post-analytical phases of laboratory operation.
2. Quality Control (QC) consists of routine, planned activities that are undertaken to ensure
accuracy and precision of all test results. This is achieved through internal quality control
procedures, external quality assurance programs and inter-laboratory comparisons.
A. Structure:
1. Organisation and leadership: The laboratory should have a proper organization
structure with assigned responsibilities, leadership, vision, and resources (finance,
human and policies) to fulfill the targets.
2. Personnel: Proper defined and documented job qualifications, job description,
orientation and training of staff, competency evaluations, provision of continuing
education opportunities should be in place. All staff should have a structured
induction training and annual competency evaluations by processing of split samples.
3. Environment and accommodation: Adequate space should be available with
segregation of activities, appropriate temperature, humidity, fire, chemical and bio-
safety programs and communication channels. In order to ensure safety, there should
be a designated laboratory safety officer whose responsibility is to train the staff and
oversee safety practices and monitor biomedical waste disposal.
C. Outcome:
Identification of nonconforming activities, root cause analysis, corrective and preventive
actions, risk assessments, complaint mechanisms, audits and monitoring of quality
indicators and management review.
All the above activities should be detailed in policy and procedure manuals such as Quality
System manual, safety manual and technical SOPs. These manuals are specific to the
laboratory and need to be written in line with local conditions.
Registration and record keeping are an integral part of the laboratory services. The basic data
regarding the names, address, sex, husband’s/ father’s name and age are registered at the point
of entry of the patient.
The STI counselors maintain a record of the individual patient in the STI card allotted after the
registration is done, which not only provides the basic data but also the medical history,
presenting signs and symptoms, findings on physical examination, laboratory investigation
done and its result, diagnosis, treatment and follow-up visits. This will allow physicians to
provide quality medical care, and will also assist in the identification of repeat offenders.
Confidentiality with regard to the results of laboratory test results is of utmost importance. Only
authorized persons can enter and access the STI card record of the patients so as to maintain the
confidentiality and a healthy patient doctor relationship. Not only is breech of confidence
punishable by law, it is also very damaging to health worker-patient relationship.
Note: The cleanliness of the slide can be judged by placing a drop of water over it and spreading
this drop. If the slide is clean and free of grease, this drop spreads into a thin even film; but if the
slide is not properly cleaned, the water collects on the slide in the form of a fine droplet and a
film cannot be made.
2. Fixing of smear
a. Heat fixing:
a) Hold the slide film upwards
b) Pass it over the flame of a Bunsen burner or a spirit lamp twice or thrice in quick
succession.
c) Feel the temperature on the back of the hand (dorsum of hand). When the slide is just
hot enough to be tolerated, fixing is complete. Too much heating chars the smear and
alters the morphology of the organisms. Less heating fails to fix the smear and it may
be washed away during the staining procedure. Fixing kills the organism, fixes it to the
slide, prevents autolytic changes, makes the organism permeable to the dye and
harmless to the person handling the smear. After heat fixation, the film is stained.
Alternatively,
Safranin powder: 10 gm
D/W: 1000ml
Make a paste of safranin powder in 50 ml D/W. Then make volume upto 1000 ml
with D/W. Filter and use.
For gonococcus Sandiford’s counterstain is useful, particularly when the organisms are few in
number. Neutral red can also be used as a counterstain for gonococcus.
Sandiford’s counterstain
Malachite green 0.05 gm
Pyronine 0.15 gm
Distilled water to 100 ml
The stain keeps for about a month.
Stain the slide in the usual manner. Instead of safranin, apply Sandiford’s counterstain for 2
minutes, flood off with water (do not wash) and blot.
Reading:
Cells and Nuclei Bluish green
Gram positive organism Purple black
Gonococci (and other Gram negative organisms) Red to pink
b. Giemsa staining
Reagents:
1) Giemsa powder 1.0 gm
Dissolve the Giemsa powder in 66 ml of Glycerol. Heat the mixture at 56 deg C for 90-120
mins. Cool it to room temperature. Add 66 ml of Methanol and mix thoroughly and allow
to stand for 7 days at room temperature (or 37 deg C). Filter. It is now ready for use after
making a working solution with buffer.
b) Na H2PO4 9.2 gm
Distilled water 1000 ml
Procedure:
Rapid Method:
1) Take the methanol fixed smear and stain with 1 part stain and 9 part buffer solution for
30 minutes.
2) Wash the slide with buffer for about 30 second.
3) Blot the slide and allow to dry in air.
Slow Method:
1) Take the methanol fixed smear on slide and place it in a Petri dish with one end on a glass
rod, film downwards.
2) Mix 1 ml of the stain with 20 ml of diluent buffer and pour into the Petri dish so that there is
sufficient stain between the film and the bottom of the Petri dish.
3) Leave the slide in the stain overnight (16-24 hours).
4) Wash the slide with buffer, allow it to dry in the air, mount and observe under oil
immersion lens of the microscope.
c. Papanicolaou Staining
Reagent:
Fixative: Equal parts of 95 % alcohol and ether.
50 % Alcohol : Diluted 50 ml of 95 % alcohol to 95 ml with distilled water.
70 % Alcohol : Diluted 70 ml of 95 % alcohol to 95 ml with distilled water.
80 % Alcohol : Diluted 80 ml of 95 % alcohol to 95 ml with distilled water.
100 % Alcohol : Mix Absolute alcohol with pulverized and previously dried Copper
sulphate (CuSO4). The Copper Sulphate settles to the bottom of the
bottle and 100 % alcohol remains above.
Harris Haematoxylin: Recommended here as a nuclear stain.
Haematoxylin: crystal 1 gm
95 % alcohol 10 ml
Ammoniuim or potassium alum 20 gm
Distilled water 200 ml
Orange G:
Dissolve 0.5 gms of Orange G in 100 ml of 95 % of alcohol and add 0.015 gms of
phosphotungstic acid ( if Orange G 6 is used; if Orange G 8 is used, add 0.010 gms of the acid).
Stain EA 36:
0.5 % alcoholic light Green SF Yellow 45 ml
0.5 % alcoholic Bismarck brown 10 ml
0.5% alcoholic eosin yellow 45 ml
Phosphotungstic acid 0.2 gms
Saturated aqueous solution of lithium carbonate 1 drop
Stain EA 25:
0.5 % alcoholic light Green SF Yellow 44 ml
0.5 % alcoholic Bismarck brown 12 ml
0.5% alcoholic eosin yellow 44 ml
Phosphotungstic acid 0.17 gms
Saturated aqueous solution of lithium carbonate 1 drop
Mounting medium : Canada balsam
Procedure:
Make 0.5 % alcoholic solution first. Because the solubility of the dyes in 95 % alcohol is low, heat
the solution at the time of preparation. Keep the solution in stock without filtering. Filter stains
EA 36 and 25 to remove undissolved stain particles.
1) Fix smears immediately (before drying) in equal parts of alcohol and ether for 5 to 10 minutes
2) Carry out the staining process in Coplin jars or staining dishes.
3) After fixation, transfer the slides immediately, without drying, directly from the alcohol
–ether mixture to 80 % alcohol.
4) Dip the slide 4 times in the coplin jar with 80 % alcohol.
5) Dip the slide 4 times in the coplin jar with 70 % alcohol.
6) Dip the slide 4 times in the coplin jar with 50 % alcohol.
7) Dip the slide 4 times in the coplin jar with distilled water.
Rapid-Diff Staining.
Reagents:
1. Solution A Fixing Solutions: Thiazine Dye in Methanol
2. Solution B Acid Dye: Eosin Y in Phosphate Buffer
3. Solution C Basic Dye: Thiazine Dye Mixture
Procedure:
1. Prepare the smears on grease free slide, and air dry.
2. Place the smear in RapiDiff fixative solution A (RapiDiff fixative) for 15 seconds (6 dips).
3. Transfer, without rinsing or drying, to solution B (Eosin Y) and stain slowly agitating the
slide in the solution or immersing and withdrawing the slide several times during for 15
seconds period. Drain excess stain onto absorbent paper.
4. Transfer slide to solution C (Thiazine Dye Mixture) and stain as slowly agitating the slide in the
solution or immersing and withdrawing the slide several times during for 15 seconds period.
5. Finally rinse the slide in Phosphate buffer pH 6 8.
6. Air dry the stained slide and examine by light microscopy under oil immersion (1000 x
Magnification).
Procedure:
1) Suspend the chemical salts, agar and charcoal in 1000 ml of distilled water.
2) Boil to dissolve the agar completely.
3) Check the pH (7.2).
4) Distribute into bijou bottles filling them completely. Keep stirring during distribution
(This keeps the charcoal uniformly suspended).
5) Sterilize by autoclaving at 121°C for 15 minutes.
6) Cool immediately in cold water (This keeps the charcoal uniformly suspended).
7) During cooling, keep inverting the bottles frequently (This again helps in uniform
suspension of charcoal).
Ingredients:
Sodium thioglycollate 1.0 gm.
Sodium glycerophosphate 10 gm.
Calcium Chloride 0.1 gm.
Methylene blue 0.002 gm.
Agar 3.0-5.0 gm.
Distilled water 1000 ml.
3) CHOCOLATE AGAR
Ingredients:
Columbia agar base 44 gm.
Distilled water 1000 ml.
Horse blood/Sheep blood 90 ml/100 ml.
Ingredients:
1) GC agar base 36 gm
2) Saponin lysed Horse blood/Sheep blood 90 ml (9% final conc.)
3) Distilled water 1000 ml.
4) VCNT inhibitor 10ml
i) Vancomycin 3.0 mg or lincomycin 1 mg/litre
ii) Colistin 300,000 units/7.5 mg/litre
iii) Nystatin 12.5 IU/ml
iv) Trimethoprim 2mg./litre.
10% SAPONIN
10% saponin is prepared by dissolving 5 gm saponin in 50 ml distilled water. Sterilize by
autoclaving at 121° C for 15 minutes.
Procedure:
1) Mix the salts and indicator in distilled water.
2) Adjust the pH to 7.1 to 7.2.
3) Autoclave/Sterilize by Seitz filter.
O
4) Store in the refrigerator at 4 C.
Chlamydia trachomatis
2) Supplements:
Foetal Calf serum 10 ml
Gentamicin 10 mg
Vancomycin 10 mg
Amphotericin B 0.5 mg
1) ENRICHED GC AGAR
Procedure:
1) Dissolve 36 gms of GC base in 450 ml of distilled water.
2) Boil the mixture.
3) Dissolve 10 gm of haemoglobin in 500 ml of distilled water separately.
4) Boil the mixture.
O
5) Autoclave both the mixtures at 115 C for 20 minutes.
O
6) Cool to 50-55 C.
7) Mix 10 ml of Isovitalex or CVA solution, 3 ml of 100 mg % Vancomycin solution and 50
ml of foetal calf serum with the GC base under strict aseptic precautions.
8) Mix the hemoglobin solution with the GC medium.
9) Mix and pour 14 ml of medium in a Petri dish of 90 mm diameter.
2) 5 % Horse blood.
3) 1 % Isovitalex or CVA (refer Enriched GC Agar)
4) 5 % Foetal Calf Serum (FCS).
5) 3 mg Vancomycin per litre.
Procedure:
1) Dissolve 38 gm of Mueller Hinton agar in 900 ml of distilled water.
2) Boil the mixture.
O
3) Autoclave the mixture at 115 C for 20 minutes.
O
4) Cool the solution in water bath to 80 C.
O
5) Add 50 ml of horse blood, mix thoroughly and immediately cool to 50-55 C.
6) Mix 10 ml Isovitalex or CVA solution, 3 ml of 100 mg % vancomycin solution and 50 ml
of FCS with this solution.
7) Mix well and pour 14 ml of medium in sterile Petri dish of 90 mm diameter under strict
aseptic precaution.
8) Pour multiples plates and store in refrigerator after the medium cools.
3) PHOSPHATE BUFFER
Solution B
Na2HPO4. 2H2O 4.46 gm
Distilled water 1000 mnl.
Prepare 100 ml of phosphate buffer by mixing 50.8 ml of solution A and 49.2 ml of solution B.
Ingredients:
1) Mueller Hinton II agar (formula per litre)
Beef extract 2.0 gm
Acid hydrolysate of casein 17.5 gm
Starch 1.5 gm
Agar 17.0 gm
Procedure:
Prepare the complete medium in a similar fashion as explained in Enriched Mueller Hinton
Chocolate Agar.
Procedure:
1) Mix 30 gm of solid with 1000 ml of distilled water.
2) Distribute an amount of 2 ml in small tubes.
3) Close the tube with cotton stopper.
4) Autoclave at 115O C for 20 min.
5) Store in refrigerator after the tubes cool down.
Stock solution A
1. Sodium chloride 160 gms
Potassium chloride 8 gms
Magnesium sulphate 2 gms
Magnesium chloride 2 gms
Distilled water 800 ml
Stock Solution B
Disodium hydrogen phosphate 3.04 gms
Potassium di hydrogen phosphate 1.2 gms
Glucose 20 gms
Distilled water 800 ml
1) Dissolve the chemical in distilled water.
2) Add 100 ml of 0.4 percent phenol red in NaOH.
3) Make the final volume to 1000 ml with distilled water.
4) Store at 4O C after adding 2 ml chloroform.
For Use
Stock solution A 100 ml
Stock solution B 100 ml
Distilled water 800 ml
Candida spp.
1) SABOURAUD’S DEXTROSE AGAR
Ingredients:
Dextrose 40.0 gm
Neopeptone 10.0 gm
Agar 15.0 gm
Distilled water 1000 ml
Procedure:
1) Boil the cornmeal in 1 litre of distilled water for 60 minutes.
2) Filter through a filter paper or muslin.
3) Add the agar powder to the filtered solution.
4) Dissolve the agar by heating in a steam sterilizer.
5) Sterilize by autoclaving at 115OC for 20 minutes.
Inositol 2-4 mg
Dextrose 3-6 mg
Maltose 5-7 mg
Raffinose 10-14 mg
Cellobiose 10-14 mg
Melibiose 15-20 mg
Procedure:
1) Make 100 mg/ml stock solutions of the sugars.
2) Sterilize by Seitz Filtration and store in sterile bottles with droppers.
3) Find the number of drops delivered per ml of the solution. This gives the amount of
sugar delivered per drop.
4) Accordingly place the requisite number of drops in each quadrant.
e.g 1 drop = 0.05 ml of volume.
Melibiose stock solution has a concentration of 100 mg/ml. So one drop will have to be
superimposed on the same spot in the particular quadrant of the plate to give the
concentration of 16-20 mgs melibiose/quadrant.
5) Alternatively, prepare the stock solution in such a concentration that each drop gives
the required milligrams of the carbohydrate.
e.g 1 drop = 0.05 ml.
Melibiose will have to be made in a concentration of 400 mg/ml because then 1 drop
will deliver 400 mg/ml x 0.05 ml = 20 mg to the single test quadrant.
Trichomonas vaginalis
Procedure:
1) Mix the contents in distilled water.
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2) Autoclave the medium at 115 C for 20 minutes.
O
3) Cool the medium to 50 C and add 100 ml sheep or bovine serum (sterilized by
filtration) and 10 mgs % Chloramphenicol.
4) Store at 4OC till use.
Ingredients:
1) Kupferberg trichomonas broth base medium
2) Beef or human serum
3) Trichomonas selective supplement
Procedure:
Suspend 23.50 gm of Kupferberg trichomonas broth base medium in 950 ml of distilled water.
O
Boil to dissolve the medium completely. Sterilize by autoclaving at 121 C, 15 lb. pressure for 15
O
minutes. Cool to 50-55 C and aseptically add 50 ml of sterile beef or human serum and
rehydrated contents of Trichomonas selective supplement.
Ingredients:
1) Dextrose 5.0 gm
2) Sodium chloride 6.5 gm
3) Proteolysed liver 2.0 gm
4) Distilled water 1000 ml (pH 6.5)
Procedure:
Mix 5 gm of dextrose, 6.5 gm of Sodium chloride and 2 gm of proteolysed liver in one litre of
O O
distilled water. Sterilize by autoclaving at 121 C, 15 lb. pressure for 15 minutes. Cool to 50-55 C
and aseptically add 80 ml of horse serum, 1 gm of benzyl penicillin and 5 gm of streptomycin.
Pour 5 ml medium into each tube.
5) TYM MEDIUM
20 gms Trypticase (casein digest peptone)
10 gms Yeast extract
5 gms Maltose
1 gm L cysteine hydrochloride
0.2 gms Ascorbic acid
0.8 gms Potassium phosphate dibasic
0.8 gms Potassium phosphate monobasic
1. Dissolve the above in 600ml Distilled water
2. Adjust pH to 6
3. Bring to 900 ml of Distilled water
4. Dispense in 90 ml amounts
5. Autoclave for 15 minutes at 121°C under 15 lb/square inch
6. On cooling add 10 ml of heat inactivate of bovine serum
7. Can be stored at -20°C
8. To improve isolation, add 0.5 gm of Bacto agar to the medium.
8. TY1-S 33 MEDIUM
} 1 gm Potassium phosphate dibasic
} 0.6gm Potassium phosphate monobasic
} 20 gmsTrypticase (casein digest peptone)
} 10 gms Yeast extract
} 10 gmsGlucose
} 1 gm L cysteine hydrochloride
} 0.2 gms Ascorbic acid
} 1 ml Ferric ammonium citrate
1. Dissolve the above in 600ml Distilled water
2. Bring to 880 ml of Distilled water
STANDARDS FOR STI / RTI LABORATORY SERVICES AT REGIONAL STI CENTRES (RSC)/ STATE
REFERENCE CENTRES (SRC)
i. Location
State: ..................................................................................................................................
District: ...............................................................................................................................
Name of Centre: .................................................................................................................
Complete address: ..............................................................................................................
Name and Phone Number of Service Provider: ...................................................................
Unique ID Number : ............................................................................................................
CHECK LIST
1. Laboratory performance
2. Laboratory staffing
3. Microbiologist performance
4. Lab.technician performance
5. Clinician performance
6. Counsellor performance
The STI/RTI Clinics in the periphery should be equipped to perform following simple diagnostic
procedure:
BACTERIOLOGY
Introduction
The isolation and identification of bacteria causing sexually transmitted infections is
accomplished by several techniques in the laboratory. A single test is often inconclusive and
hence, many tests may have to be performed in order to arrive at a correct diagnosis. These
techniques include-
a. Direct examination of bacterial smears- By wet mounts or stained smears. This is generally
not sufficient to identify the bacterial species, although it can give a fair idea about the
type of bacteria to expect and media to be used for culture. This shows the importance of
proper collection and dispatch of specimens to referral laboratories. Nevertheless, direct
microscopic examination of stained smears is an efficient way of studying the presence of
bacteria in biological fluids that are normally sterile, such as cerebrospinal fluid (CSF) and
pleural fluid, and in specimens from other sources. It may provide information of great
value for the diagnosis, immediate treatment and control of the disease in limited cases.
For eg.- -
I. The value of direct smears may be high in specimens from cases of male urethritis, as
an early stage gonococcal infection can be diagnosed with reasonable certainty (in
females it is much more difficult).
b. Culture of bacteria in artificial media- When conditions appropriate to the growth of
bacteria and similar to the human body milieu can be provided in the laboratory, in the
form of artificial media in Petri dish or test tubes, then it is possible to isolate the bacteria
in the laboratory. Eg. It is possible to culture Neisseria gonorrhoeae and Haemophilus
ducreyi in the laboratory.
c. Biochemical tests on the isolated cultures- Performing certain biochemical tests will often
bring out certain unique properties of bacteria and thus aid in the accurate species level
diagnosis.
d. Serological tests- The diagnosis of some diseases is also possible through serology,
especially when culture of the bacteria is not feasible. Eg. Syphilis. Serological techniques
are also important for sero epidemiological surveillance.
Principle
The sample to be examined (pus, urethral/cervical/vaginal discharges, urine centrifugate,
cerebrospinal fluid, etc.) is treated as follows:
} It is spread in a thin layer on a glass slide
} It is dried completely
} It is fixed on the slide by gentle heating before being stained.
Inoculating loop
Inoculating loop
This is a metal wire (usually made of nickel-chromium
alloy-Nichrome) fixed on to a handle and bent into a
loop at the other end. Make the loop with forceps,
taking care that it is centred.
Preparation of smear
Hold the loop just above the blue part of the flame
Hold the instrument as nearly vertical as possible.
Flame the loop until it is red-hot
Allow to cool (count to 20)
Figure 4. Flaming
Place the loop on the slide and press slightly flat and
in the center (the slide should be numbered) (Fig.7).
Make a smear using a swab or loop.
Let the slide dry completely in air. Label the slide with
a glass maker pen/ pencil. (Fig.9)
Fixation
The smear has to be fixed on the slide. (Fig 11). Check
that the smear is completely air-dried
Pass the slide through the flame of a Bunsen burner,
with the smear uppermost. Pass it through the flame
three times in quick succession. Allow to cool before
staining.
Figure 11.Fixing of smear.
It is sometimes useful to draw a circle around the
smear with a grease pencil, so that it can be seen more easily.
Gram Staining
Gram stain- is a differential staining technique, described
first by Hans Christian Gram in 1884.
Figure 12. Staining reagents
Advantages-
Gram staining makes it possible to classify bacteria into two groups:
1. Gram positive bacteria- stain dark violet.
2. Gram negative bacteria- stain pink.
This makes identification easier.
Gram staining reagents :
Primary stain- Crystal violet/Gentian violet
Gram’s iodine, solution 95% ethanol, Safranine solution and tap water.
Technique:
Fix the smear and allow cooling. Crystal violet- Pour the crystal violet on to the slide. Cover the
slide completely. Leave for 1 minute. Rinse with tap water and drain.
Gram’s iodine solution – flood the slide with Gram’s iodine solution. Let it stand for 1 minute.
Drain off the solution and rinse with tap water.
Draining
Drain and allow to dry in the air. (Fig.14)
QUALITY CONTROL
A known gram positive and a gram negative stain should be used as quality control;
S. No. Control Organism Expected Result
1 S.aureus ATCC 25923 Gram positive cocci
2 E.coli ATCC 25922 Gram negative bacilli
Peudomonas aeruginosa ATCC 27853
Bacteria are 0.1- 0.5u x 3-7 u in size and can only be visualized under a microscope or isolated in
artificial culture media when they form colonies.
A distinction is made between the following types of bacteria-
Pathogenic bacteria*
These are capable of causing disease in man. They are found in the body they infect and are
detected by laboratory methods.
Non-pathogenic bacteria
These are harmless bacteria that exist in countless numbers in nature. Some multiply normally
in man without affecting his health and are known as commensals. Others are capable of
causing infection and disease when the opportunity arises (like underlying diseases or immuno
compromised status of host). Such bacteria are called Opportunistic pathogens.
Direct examination is most useful in obtaining an indication of the type of organism involved or,
in some cases, in establishing a diagnosis of the disease. Eg. Gonorrhoea. For this purpose it is
essential to give a detailed description of the organisms seen as well as of any other elements
present (leukocyte, red blood cells, epithelial cells etc.)
Figure 20 Candida
Figure 21 Actinomyces
In healthy persons the following are sterile: In healthy persons following may contain
} Blood many non-pathogenic organisms
} Cerebrospinal fluid (commensals).
} Subcutaneous tissue } Respiratory tract (mouth, nose, throat,
} Internal organs (heart, liver, kidney, etc.) (sputum)
} Gastrointestinal tract
} Skin, ear and eye
} Urogenital tract (vagina, anterior
urethra)
In disease all of these areas may be infected by pathogenic organisms.
Elements
Type: leukocytes, red blood cells, epithelial cells.
Organisms:
Shape cocci, bacilli, etc
Arrangement pairs, chains, clusters
Staining properties Gram, Ziehl-Neelsen
Special Characteristics Spores, granules, etc
Quantity occasional, a few, a moderate number, many, plenty, profuse
Example of reports
1) Pus from abscess (direct bacteriological examination) (Gram stain):
} Many leukocytes
} A few red blood cells
} A few epithelial cells
} A moderate number of Gram positive cocci in clusters
2) Urine (direct bacteriological examination) (Gram stain):
} A few leukocytes
} Occasional red blood cells
} A few epithelial cells
} A few Gram negative bacilli
3) Sputum (direct bacteriological examination (Ziehl-Neelsen)
} 5 acid- fast bacilli found/10 fields (2+)
Important:
It is rare to diagnose a disease in the laboratory on the basis of the identification of the
organisms found by direct bacteriological examination of a specimen. The results of such an
examination, however, can help the physician to establish a diagnosis when taken together with
the symptoms shown by the patient.
GONORRHOEA
This is one of the important STIs caused by Neisseria gonorrhoeae. The incubation period is 4 to
8 days. Typical symptoms include genitourinary discharge. Genitourinary discharges may occur
in other diseases too, as mentioned below:
Thick yellow pus: Likely to be Gonorrhoea
Clear whitish fluid, frothy and foul smelling: Trichomoniasis
Thick, curd-like, cheesy-white exudate: Fungus
Other exudates: Non - specific urethritis (not identifiable by direct
examination).
Principle -
Smears of urethral pus are stained with Gram stain. Gonococci can be recognized by three
characteristics:
1. Diplococci (in pairs)
2. Gram negative
3. Intracellular (inside the leukocytes).
Examination of slides-
Pay particular attention to the edges of the smear, where the elements are spread more thinly
and are easier to see and the stain is less concentrated.
Pus (Note whether there are many masses of degenerate leukocytes. The nuclei are bright pink
and the cytoplasm is colorless).
Gonococci oval, kidney-shaped, Gram negative (pale pink) cocci
A few red blood cells A few red blood cells A few red blood cells
A few epithelial cells A few epithelial cells A few epithelial cells
A moderate number of gram No gram negative No gram negative
negative intracellular Intracellular diplococci. Intracellular diplococci
diplococci Gram negative extracellular Extracellular diplococci
diplococci seen.
CONCLUSION
Gonococci-Positive Gonococci-suspicion Gonococci-negative
Male: A number of the following may occasionally be seen in smears of urethral pus:
} Gram positive cocci (Staphylococci)
} Gram positive bacilli (Diphtheroids)
1. Place the bottle upright. Collect the pus specimen on a swab. Unscrew the bottle cap.
2. Holding the bottle as upright as possible (to prevent the gas escaping), rub the swab of pus
over the whole surface of the solid medium, from one side of the bottle to the other, from
the bottom. (Fig.26)
3. Replace the cap on the bottle at once.
4. Dispatch at normal temperature.
Preservation time: up to 3 days, but the shorter the delay, the better.
SYPHILIS
Syphilis is another important venereal disease, caused by a spirochaete: the pale treponeme
(Treponema pallidum).
The first sign of the disease appears, usually on the genital organs as a chancre, round or oval, 1
or 2 cm in diameter, red, with firm, indurated edges.
Non -venereal (endemic) syphilis
This form of the disease is found in semi-desert regions such as the Sahel belt south of the
Sahara and the Eastern Mediterranean area. It mainly affects children.
YAWS
This is a non-venereal disease found in humid tropical climates.
It is caused by a different treponeme (T. pertenue), which looks identical to T. pallidum.
Direct wet examination for treponemes in syphilis and yaws-
This can be carried out only by experienced personnel in a laboratory equipped with a dark field
condenser microscope (Dark field microscope).
The examination is of no value when the patient has treated the lesion with ointment. In that
case, wait 3 days before making the examination.
1. Clean the chancre with gauze moistened with sterile sodium chloride solution. Dry it.
2. Scrape the edges of the chancre several times with the flat blade of a sterile lancet. Do
not draw blood.
3. Press with dry sterile gauze.
4. Remove the swab and wait a few minutes until a pinkish serous fluid appears.
Draw off the fluid with a Pasteur pipette with a teat.
5. Place a drop of the fluid on a thin glass side (designed especially for dark ground
microscopy).
6. Examine under the microscope using a dark field condenser.
The treponemes of syphilis and yaws can be distinguished from saprophytic treponemes of the
skin by their very thin bodies and characteristic movement.
The technician needs special training to recognize them.
Examination of dried and stained smears- This is not recommended because of the presence of
saprophytic treponemes on the skin and in the mucous membranes.
URINE EXAMINATION
Urinary Deposits
Principle
Urine contains microscopic elements in suspension (cells, crystals, etc). These elements are
collected by centrifuging and a drop of the deposit is examined between a slide and coverslip. As
all these elements in suspension would sediment in the urine if left for a few hours, they are
called urinary deposits.
Collection of urine
Examine a specimen passed in a single urination.
Examine a mid-stream specimen of fresh urine as soon as possible; it should be
} Collected in the laboratory
} Or brought quickly from the patient’s room (within 2 hours of voiding)
} The sterile receptacle should be provided by the laboratory
} Women should be instructed to wash the genitalia before hand
} Men should collect urine sample after cleaning and retracting the prepuce
} Never carry out the examination on urine kept in the refrigerator
Value
In certain diseases of the urinary tract the urinary deposits are considerably altered. The
following elements may be found:
} Pus cells
} An abnormal number of red blood cells (RBC)
} Parasitic forms
} Casts
Preservation of urine – can be done with formaldehyde solution, by adding 8 drops of 10%
formaldehyde solution per 300ml of urine.
Note - Urine treated in this way cannot be used for any other laboratory test.
Materials
} Table top centrifuge
} 15ml conical centrifuge tube
} Capillary dropping pipette (Pasteur pipette), if possible calibrated, to deliver 50 drops
per ml
} Slide and coverslip, 20 X20 mm
} If necessary, 10% formaldehyde solution
How to express the quantity of red and white cells found in urine deposits-
It is important to mention the quantity of the various elements found. It is important always to
use the same method of expressing quantities found.
With
1 drop of urine deposit (1/50 ml)
1 coverslip, 20X20 mm
X 40 objective; eyepiece x5 or x6
Examine microscopically
Red Cells
0-10 red cells per field - few red cells (normal)
10-30 red cells per field - moderate number of red cells
Over 30 red cells per field - many red cells
Leukocytes
0-10 leukocytes per field - few leukocytes (normal)
10-20 leukocytes per field - moderate number of WBC
20-30 leukocytes per field - many leukocytes
Clumps of more than 20 degenerate leukocytes - many leukocytes seen in clumps
Clumps and many degenerate Leukocytes - full field
C. Yeasts
Do not confuse with red cells
Size - 5-12µm
Shape - round or oval bodies of various sizes found together
Budding may be seen
They are not soluble in acetic acid.
Yeasts are occasionally present in urine containing glucose. Check that the urine is fresh.
D. Trichomonas vaginalis
Size - 15µm
Shape - round, globular
Motility - motile in fresh urine (they whirl and turn)
Undulating membrane - on one side
Flagella - 4 flagella, more or less visible
F. Epithelial cells
1. Squamous epithelial cells- Large rectangular cells, the product of desquamation
(the shedding of cells from the epithelium of the urinary tract and organs). They
come from:
- The ureter or
- The vagina
2. Bladder cells- Large cells often diamond shaped with a distinct nucleus
3. Cells from pelvis of the kidney- Medium sized cells (the size of 3 leukocytes)
granular, with a sort of tail
4. Cells from the ureter and pelvis of the kidney. Medium sized oval cells with a distinct
nucleus. If many are present together with leukocytes and filaments, they may be
from the ureter. If few present, with no leukocytes, they may be pelvic cells.
5. Renal cells- Renal cells are small. They are the size of 1-2 leukocytes, very granular.
The nucleus is refractile and clearly visible. They are almost always present with
protein in the urine.
G. Casts: Casts are cylindrical in shape and long, crossing almost the whole field when
examined under the 40x objective. They are formed during disease in the renal tubules, which
may fill with blood and other cells and chemical deposits.
1. Hyaline casts: Transparent and slightly retractile, the ends rounded or tapered.
(They may be found in healthy persons after strenuous muscular effort).
2. Granular Casts: Rather short casts filled with large granules pale yellow in color, with
rounded ends (The granules come from degenerate epithelial cells from the tubules
of the kidney) .
3. Fine granular casts: The granules are smaller and do not fill the cast. Do not confuse
with hyaline casts partly covered by amorphous phosphate crystals.
4. Blood Casts- Casts filled with more or less degenerate red blood cells, brownish in color.
5. Pus Casts: Casts filled with degenerate leukocytes. True pus casts are completely
filled with leukocytes. Hyaline casts may contain a few leukocytes.
6. Epithelial casts : Casts filled with pale yellow epithelial cells (to make the cells more
distinct, add a drop of 100 g/l (10%) acetic acid to the deposit)
7. Fatty casts(rare) : Very refractile yellowish casts, the edges indented and distinct,
the ends rounded. Fatty casts are soluble in ether but not in acetic acid. (They are
found in cases of severe kidney disease).
I. Crystals:
Crystals have regular geometric shapes unlike amorphous debris, which is made up of clumps of
small granules with no definite shape.
B. AMORPHOUS DEBRIS
1. Amorphous phosphates (alkaline urine)
Granules small, whitish, often scattered
They are soluble in 100 g/l acetic acid (1 drop per drop of deposit)
2. Amorphous urates(acid urine) : Granules very small, yellowish, grouped in compact
clusters. They are not soluble in 100 g/l acetic acid but dissolve if the urine is gently
heated. [Urine kept in the refrigerator often shows a heavy precipitate of urates]
3. Dr. Meenakshi
Microbiologist
Regional STD Centre
Safdarjung Hospital & V.M.M.C.
New Delhi
8. Dr Hema Kapoor
Department of Microbiology
Safdarjung Hospital, New Delhi
6th & 9th Floor, Chandralok Building, 36 Janpath, New Delhi - 110 001, India
Phone: 91-11-4350 9999 / 2373 1774