1734601443372

King Khalid University Hospital
King Saud University

Applied Medical Sciences
Clinical Laboratory Sciences
Bacteriology
Raghad Obaid Alamri
Bacteriology lab include different benches; Stool bench, Urine

bench, Blood bench, Respiratory bench and General bench.
In general, the main tests the lab specialist must follow are;
-Cultivation(Growth).
-Identification and Antimicrobial testing.
-Direct detection of microorganisms by microscope.
-Direct detection of specific product produced by the infected
organisms using chemical, immunological or molecular techniques.
Safety precautions:
-Must handle the specimens with proper personal protective
equipment (PPE).
-Specimens should be opened in a Class-II biological

safety cabinet.
NOTE: Minimize the movement when using the
biosafety cabinet since its disrupt the airflow.
Clean the cabinet with disinfectant after use.
-Practice proper hand washing before leaving the lab.
Notify the supervisor immediately for any accidents
or unsafe conditions.
In all microbiology labs, the workflow for identification of

bacteria and fungi are the same.
Gram stain → Cultivation → Biochemical tests.
1- Gram stain:
The structure of the organism's cell wall determines whether it's
gram positive or gram negative.
When stained with a primary stain and fixed, some bacteria are able
to retain the primary stain by resisting decolorization while other get
decolorized by decolorizer.
Bacteria that retain the primary stain called Gram positive.
Bacteria that get decolorized then get counterstained called Gram
negative.
Method:
2-Cultivation techniques:
Culter media
Blood agar
Non-selective (support both gram +ve and -ve growth).
Differential (β-hemolysis, α-hemolysis or γ-hemolysis ).
MacConkey agar
Selective and differential for gram -ve bacteria.
Differentiate between lactose fermenters from Non.
Contain bile salts and crystal violet to inhibit gram +ve bacteria.
Chocolate agar
Contain X and V factors required for H. Influenzas growth.
Xylose Lysine Deoxycholate agar (XLD)
Differentiate and isolate Shigella (Red colonies) and Salmonella spp
(Red colonies with black center) from other enteric bacteria.
Coliforms will give Yellow to Orange colonies. (Non-significant)
Hekaton Enteric agar(HE)
Differentiate and isolate Shigella (Green colonies) and Salmonella
spp (Green colonies with black center) from other enteric bacteria.
Coliforms will give Yellow to Orange colonies. (Non-significant)
Cystine Lactose Electrolyte Deficient agar (CLED)
Non-selective, used to differentiate and isolate urinary microbes.
Electrolyte deficient prevent the swarming of Proteus sp.
Bromothymol blue is the indicator, change into yellow in case of acid
production from lactose fermentation.
Columbia blood Agar
Selective media with antimicrobial additives, used for the isolation of
anaerobic Gram +ve organisms.
Campylobacter Selective Agar (CAMPY)
Enriched blood medium, selective for Campylobacter jejuni subsp.
Inhibit the growth of normal flora.
Yersinia Agar (CIN)
Selective medium used for the isolation and differentiation of Yersinia
enterocolitica sp.
Mannitol salt agar (MSA)
Selective medium for the isolation of pathogenic staphylococci spp
(S.aureus & S.epidermidis).
Contains phenol red as a pH indicator, changes into yellow in case of
mannitol fermentation.
Sabouraud dextrose agar
Non-selective for isolation of pathogenic and non-pathogenic fungi.
Modified Thayer-Martin agar (MTM II)

Selective media for the isolation of pathogenic Neisseria spices.
Protocols Phenylethyl Alcohol agar (PEA)
Selective medium used for the isolation of gram +ve Staphylococcus
species and Streptococcus species.
Other special medium

Robertson's cooked meat broth (RCM)
Enrichment for all kinds of bacteria, favorable for Anaerobes.
Contain cooked meat heart muscles, these muscle proteins and heart
tissue granules are a source of amino acids and other nutrients.
Growth is indicated by turbidity and gas bubbles with some organisms.
Disintegration and blackening of the meat particles indicates
Proteolysis.
Selenite broth
Selective for the Enrichment of salmonella species.
Growth is indicated by turbidity.
Not enough for diagnosis. Must be sub-cultured in an agar such as HE.
Todd Hewitt broth (TH)

Enriched media used selectively for the growth of streptococcus species
especially Enterococcus sp to fasten the growth because it's used for
clinically important samples like CSF.
Growth is indicated by turbidity.
Blood culture system

A broth-based medium that supports the growth of aerobic and
anaerobic bacteria, depending on the bottle type used, and also some
fungi, predominantly yeasts.
For detection bloodstream infections.
Dip slide
Used for monitoring bacteria in water systems.
3-Identification techniques:
antimicrobial susceptibility testing are important to confirm
susceptibility to chosen empirical antimicrobial agents, or to detect
resistance in individual bacterial isolates.
It determined the sensitivity and resistance of microorganisms
against anti-microbial agents and identifies the suitable antibiotics
and their dose.
It's done manually or automated.
Automated methods:
We use instruments that could provide both identification and
antimicrobial susceptibility testing.
Principles of Technologies Used:
A hands-off incubation and reading functions for microdilution trays
or special cards in an incubator-reader device.
1- VITEK®2:
One of the most accurate devices for diagnosing
bacteria and determining the appropriate
antibiotics since it measures the MIC for all types
of antibiotic specific for each type of bacteria.
Therefore, it helps in the treatment and
determining the dose.
It provides ID\AST results in about 6 hours.
Used mostly for sterile body fluids.
NOTE: Run a QC once every week.
Test cards set up:

VITEK® 2 ID and AST colorimetric reagents cards have 64 wells, each
contain an individual test substrate. Substrates measure various
metabolic activities. They provide identification and AST results for
clinically important microorganisms.
These cards are based on the broth MIC technique.
They are incubated and interpreted automatically and read

turbidimetrically every 15 min.
ID cards
GN Gram negative bacilli(LF and NLF).
GP Gram positive cocci and non-sporing bacilli.
NH For Haemophiles, Neisseria species and
microaerophilic gram negative bacilli.
ANC For Anaerobic and Coryneform bacteria.
AST cards
AST-P580 For Staphylococcus species.
AST-ST03 For Streptococcus species EXCEPT enterococcus spp.
AST-GP67 For Enterococcus spices.
AST-N91 For Lactose fermenting gram negative bacilli.
AST-N92 For pseudomonas and Acinetobacter only.
2- DxM MicroScan WalkAway ID/AST System:

The WalkAway is an automated bacterial identification
and susceptibility testing system. It uses direct
minimum inhibitory concentrations (MIC) with accurate
detection of antimicrobial resistance.
Principle:
Fluorogenic substrates are used for identification.
Hydrolysis of the substrate will cause a pH change
resulting in fluorescent signals.
How it work:
Bacterial suspension from culture is used to inoculate
into the panels.
A barcode label and the end of each panel indicates the
sorts of tests that will be run.
NOTE: Red panels are for gram -ve, and The blue panels
are for gram +ve bacteria.
Positive and
Negative controls Classical
biochemical
identification
Determination of
susceptibility and
resistance to
antibiotics
ID section → Visual or instrumental read, biotype based on positive

or negative reaction. MIC
AST section →
Results of panels status:
Green Finished
Yellow Unfinished
Red Alarm
Gray Processing
The instrument have 8 towers, each has a 12 spots for panels.

NOTE: gram -ve bacteria results are faster than gram +ve.
NOTE: Run a QC once every week.
The difference between VITEK®2 and MicroScan:
VITEK®2 MicroScan
Used mostly for sterile body sites Used mostly for non-sterile sites
Complicated and takes longer time Easy and takes less time
AST and ID are in different panels AST and ID are in the same panel
Has more antibiotics Has less antibiotics
Short incubation time Long incubation time
More accurate Less accurate
Used mostly for strep spp Used for S.aureus and Enterococcus
spp from staph only
3- VITEK® MS:
The Vitek mass spectrometry instrument works by the
MALDI-TOF principle, which is, Matrix Assisted Laser
Desorption Ionization Time Of Flight.
It works by Identification of bacteria ONLY by determining
the mass/charge ratio of the proteins present in the
bacterium cell.
Principle:
A pulsed laser beam irradiates the sample, it will result in
sample ionization due to anergy absorption.
All ions are accelerated in the same kinetic energy, enter the
flight tube and get separated according to mass and
detected at the end of the flight tube.
The bioMerieux algorithm will translate the graph into
identification.
How it works:
The sample is mixed with a suitable matrix material
and applied to a target slide.
Each target slide contains 48 sample spots devided into
3 sections of 16 sections each called Acquisition group.
Each acquisition group has a spot in the center used for QC.
NOTE: Each microbe has different QC, E. coli are
mostly used.
Manual method:
E-test:
This test measures the minimum inhibitory concentration (MIC) of an
antibiotic needed to stop the development of microorganism.
It complements the VITEK® 2 automated system platform by offering
a flexible and precise MIC solution to test additional antibiotics.
Principle:
A bacterial suspension is done from a bacterial culture and
swipe the suspension on the Mueller Hinton Agar (MHA)
using a sterile cotton swap.
NOTE: Suspension turbidity will differ depends on the
organism, mostly 0.5McF (GPB will be 1McF).
The antibiotic strip is then placed on to an inoculated agar

plate.The antibiotics will immediately release from the strip
into the agar surface.
After incubation, bacterial growth becomes visible on the

plate, and symmetrical inhibition ellipse along the strip is
seen if there is sensitivity.
NOTE: MHA with blood used for β and α hemolytic
streptococcus spp only.
ComASP™ Colistin:
Colistin is often the last option to treat infections caused by
multidrug-resistant micro-organisms.
Compact Antimicrobial Susceptibility Panel containing colistin
that allows for testing up to four samples per panel.
It is used for a quantitative determination of the minimum inhibitory
concentration (MIC) in various concentrations of the antimicrobial
agent.
Patients who still have resistance are treated with
mix antibiotic agents.
Blood bench:
Blood in normally a sterile, which mean any bacteria or fungi found
there are significant and need immediate notification.
Blood culture should requested when BSI or sepsis is suspected.
Samples:
Blood is collected in special bottles containing anticoagulant and
inhibitors for antibiotics activities.
There are different types of blood bottles depending on;
1- The age of the patient.
2- Type of bacteria(Aerobic or Anaerobic).
3- Type of machine we use for incubation(VIRTUO® or BACTEC™ FX).
BD Bactec bottles:
Blue top; for Aerobic bacteria
Purple top; for Anerobic bacteria
Silver top; for pediatrics Aerobic
BACT/ALERT bottles:
Green; for Aerobic bacteria
Orange; for Anerobic bacteria
Yellow; for pediatrics Aerobic
Volume of blood should be 5-10ml for Adults and 1-3ml for pediatrics.
The blood bottles are incubated at 36-37C° for 5 days(90% will grow in 1-2 days).
For fastidious organisms with slow growth such as; Brucella spp (strict aerobic-
zoonotic infection), incubate it for 21days to provide the best environment for
growth.
Diagnostic methods used:

We use two machines that follow the same principle, since both of
them work as an incubator and an analyzer.
 BD BACTEC™ FX Blood Culture System
 BACT/ALERT® VIRTUO®
These blood culture automated systems uses fluorescence in
detecting the CO2 releasing from the bacteria.
NOTE: The bacteria metabolize the nutrient in the
culture media bottle thus producing CO2 as a product
or use oxygen in the
medium.
Liquid Emulsion Sensors (LES) at the bottom of each
culture vial will change color when the pH changes due to
the rise in CO2 levels.
If there is a positive result (Ve+) → Red light will show
If there is a negative result(Ve-) → Green light will show
Set up:
If there is a positive result, we culture the bottle on 3 main plates;
 Blood agar
 MacConkey agar
 Chocolate agar
Aerobic Anaerobic
Blood O2 Blood O2
MacConkey Blood O2
+ +
Chocolate MacConkey
Chocolate
o Make a smear on a slide for gram stain.
Incubate the plates for 24h at 37 C°; BA and CHOC agars are
incubated with 5% CO2 environment and MAC in O2 environment.
Do a gram stain; report Immediately to the physician if the stain
results show any organism.
Reading:
Any growth in blood is Significant.
Chemical tests required:
1-Catalase test: Distinguish between: strep and staph.
→ Ve+ for Staph.
→ Ve- for Strep.
2-Coagulase test: Distinguish between staph species.

→ Ve+ for S.aureus.
→ Ve- for other Staph spp.
Devices required:
1-Maldi-Tof
2-VITEK
3-Microscan
Respiratory bench:
At this bench we look for microorganisms that infect the lower or the
upper respiratory track.
Type of samples received:
 Sputum.
 Broncho alveolar lavage(BAL).
 Bronchial aspirate.
 Sliva. (Rejected)
Knowing where the sample came from (Upper or lower) can tell us a
lot about the sample and can contribute in narrowing down the
organism.
Set up:
By using a cotton swap, take from the sample and place it on 3 main
plates. then use a sterile loop for streaking;
 Blood agar
 MacConkey agar
 Chocolate agar
NOTE: For BAL sample, take 2 of each plate. Streak Each 3 plates
with different inoculation loops sizes.
Incubate the plates for 24h or at 37 C°; Blood and CHOC agars are
incubated with 5% CO2.
Do a gram stain for microscopic examination to determine if a
sample from the upper or lower respiratory track by using:
Q-score (Quality Score):

This test help us to determine whether the sample is from the
upper(Higher epithelial cells ratio) or lower respiratory track(Higher
WBC's ratio) by seeing the ratio of WBC's to epithelia cells.
It can also provide us with how many pathogens are responsible
potentially. And it can helps determining if the sample can be
accepted(Happens mostly with saliva samples as it’s the respiratory
sample closest to the outer microbiota).
First
Measure the pus and epithelial cells.
 1-9 → +
 10-14 → ++
 15-24 → +++
 ≥ 25 epi → Reject
Second:
Look for any organism.
 Gram –ve e or +ve
 Staph or Strep
 Bacteria or yeast
NOTE: Patients under antibiotics treatment will have no organism

appear on the smear.
Q-score: points of average number of neutrophils + points of average

number of squamous cells.
The better the sample, the higher the score
Squamous epithelial cells
The value Nill or Few Moderate ≥25

scanty
Nill or 3 0 0 Rejected
WBC's scanty
Few 3 0 0 Rejected
Moderate 3 1 0 Rejected
≥25 3 2 1 Rejected
Nill=+, Few=++, Moderate=+++
Q0 → Work only if the growth is predominant.

Q1 → Work up only 1 potential pathogen if pure of predominant.
Q2 → Work up only 1 potential pathogen. Important considerations:
Q3 → Work up only 2 potential pathogens. A sample with a '+++'

epithelial cells will have a
Q4 → Work up maximum of 3 potential pathogens. Q0 score.
A sample with no epithelial

cells will have a Q3 score.
Reading:
Compare the growth with the microscopic examination results.
Potential pathogens include:
-S. aureus
-S. pneumoniae
-S. pyogenes
-Enterobacteriaceae
-Aerobic and facultative anaerobic gram negative bacilli
-M. catarrhalis
-Neisseria meningitidis
Yeast (Not potential pathogen, sub-culture on SDA and refer to

mycology lab if the growth is pure or predominant).
Reading the growth:
BA:
Non-selective enrichment media, greenish colonies are non-significant
normal flora.
Significant gram positive species:
- S. aureus
- S. pneumonae
MAC:
Selective for gram - ve, lactose fermentation help in identification.
Significant lactose fermenting species:
- Klebsiella Pneumoniae
Significant non-lactose fermenting species:
- Pseudomonas aeruginosa
CHOC:
Enrichment media, important for just H.influenza.
Devices required:
1-Maldi-Tof
2-Microscan
3-Vitek
Stool bench:
Stool culture is ordered to determine the microorganisms that
infecting the digestive track causing food born or fecal-oral illness.
 Stool samples. Most common
 Rectal swaps.
Note the appearance of the specimen (The color, consistency,
presence of blood or mucus).
NOTE: Always examine the watery and the bloodstained
specimens first
The most important bacteria that we look for are Gram –ve
(Especially Gram negative bacilli) bacteria only since all the gram
+ve bacteria are normal flora EXEPT Clostridium difficile.
The most important isolates:
-Salmonella sp
-Shigella
- Yersinia enterocolitica
- campylobacter jejuni
Set up:
By using a sterile inoculating loop, take from the stool sample and
culture it on 4 main medias:
Important considerations:
Always start with the

CAMPY agar because its
prone to contamination,
since it’s a nutrient agar.
Selenite broth
o NO gram stain for stool, because it has a lot of microbiota.

Incubate the XLD and Yersinia plates and the selenite broth for 24h
at 37 C°.
The selenite broth must then be sub-cultured on HE then incubate it
for another 24h.
The CAMPY is selective only for Campylobacter sp and it require a
special environment (Since these species are thermophilic and
microaerophilic) so we incubate them at high temperature (42 C°) in
low O2 with CO2 environment for 48h in a CAMPY jar.
NOTE: Must incubate the plates with a QC of a positive culture
of campylobacter jejuni to check if the jar environment is
efficient for growth.
A CampyGen sachet is placed into the jar, it will rapidly absorb the
atmospheric oxygen in the jar with the simultaneous generation of
carbon dioxide, producing the appropriate microaerobic conditions.
If a C. diff test is ordered:

Clostridioides difficile is considered a member of the normal gut
microflora. However, patients with prolonged antibiotic treatment
will disrupt the balance of the bacteria in the bowel, which can cause
the C. difficile to get activated and produce toxins that causes
diarrhea and colitis.
Watery stool is an important indication for testing C. diff.
C. DIFF QUIK CHEK COMPLETE ®:
A rapid membrane enzyme immunoassay for the detection of C. diff
glutamate dehydrogenase antigen and toxins A and B.
If there was a Positive Antigen result or weak Toxin result, then MUST run a PCR test to confirm.
GeneXpert® Systems:
GeneXpert Instrument Systems automate and integrate
sample purification, nucleic acid amplification, and
detection of the target sequence using real- time reverse
transcriptase PCR (RT-PCR) and real-time PCR assays.
Xpert® C. difficile:
Provides detection in 40 minutes.
Detection of three targets:
- Toxin B (tcdB)
- Binary toxin (cdtA)
- TcdC deletion
If a pathogenic E.coli (STEC O157 H7) is ordered:

If the doctor suspected STEC we culture the specimen on
S-MAC(Sorbitol MacConkey) agar. A selective and differential
medium used to differentiate pathogenic E. coli(O157 H7) from other
E. coli.
Loose and bloody stool are important indications for testing STEC.
STEC dose NOT ferment the sorbitol

therefore, it gives colorless colonies.
A- Appearance of E. coli that ferments the sorbitol.
B- Appearance of STEC that don’t ferments the

sorbitol.
Reading the growth:

24h reading:
XLD:
Selective for gram negative, Yellow colonies are non-significant
normal flora(coliforms).
All gram negative bacteria ferment the xylose EXCEPT Shigella spp
which will appear as Red colonies without H2S production.
Salmonella spp ferment the xylose BUT it utilize the lysin and gives
alkaline product so it appears as Red colonies with H2S production.
Yersinia:
Selective and differential medium used to isolate Yersinia
enterocolitica.
Growth appear as White colonies with red center.
Pink colonies are Non-significant.
Selenite broth:
Selective for enrichment of Salmonella spp.
Growth indicated by turbidity.
Not enough for diagnosis, MUST be sub-cultured in HE agar.
48h reading:
HE:
Selective and differential for Salmonella and Shigella spp.
Orange colonies are non-significant normal flora(Coliforms).
Salmonella spp appears as Green colonies with H2S production.
Shigella spp appears as Greenish blue colonies without H2S
production.
CAMPY:
Selective for Campylobacter jejuni.
Inhibit the growth of normal flora.
Growth appear as Grey glistening colonies.
Devices required:
1-Maldi-Tof
2-Microscan
3-Vitek
General bench:
This bench receive different types of specimens from different body
sites.
 Tissues
 Body fluids
 Swaps
 Catheters
 Water from machines
Set up will be different depends on the specimen type;
Set up:
1- Tissue specimens:
Tissue specimens must be grinded with saline first before start
processing.
Grind the tissue until it become a much-like and process it like a
body fluid sample.
After grinding, use a sterile loop to take from the grinded specimen
and culture it on 6 medias:
Important considerations:
GN and Columbia agars are

Anaerobic selective medias for
isolation of gram +ve and – ve
bacteria.
GN→ isolation of gram –ve

GN
Columbia→ isolation of gram +ve
Columbia
2- Body fluid specimens:

For all body fluids(Sterile and Non- sterile) important steps must be
done first before start culturing.
Set up:
First:
1-Macroscopic examination (Clear, Turbid or clotted).
2-Cell count (Count RBC's and WBC's).
NOTE: Don’t do cell count for a clotted sample.
3-Gram stain (To see if there is any organisms).
4- Differential stain (To differentiate neutrophils from monocytes).
NOTE: Done ONLY if there is high number of WBC's in cell
count.
Second:
For CSF, culture on 5 media:
CMB TH
For other body fluids, culture on (BA, MAC, CHO and CMB) only.

3-Swaps specimens:
Types of swap samples:
 HVS
 MRSA
 Wound
 Groub B
 Throat
 Bacterial vaginosis
 Eye and Ear
Set up:
Process of each sample differ depending on the site of swap
HVS swap MRSA swap
Bi plate, BA and MSA
Sabroud
Thayer martin
Wound swap

Ear and Eye swap Streps group B

Culture on BA only.
Throat swap
Bacterial vaginosis
Do a Gram stain only.
Incubate the MAC, SDA and the Screening plate at 37 C° in O2

environment and the BA,CHOC and CMB in 5%CO2 environment.
Reading the growth:

We read the growth twice, 24h and 48h reading.
Screening:
On MSA:
Differential media, S.aureus will ferment the mannitol and give diffused
Yellow color.
On BA:
Non-selective and differential media. Growth indicated by β-hemolysis
with shiny golden big colonies.
Growth in Swaps samples:

1-Veginal Swaps:
SDA:
Selective media for yeast growth.
Growth appears as white creamy colonies.
Refer to mycology lab.
TM:
Selective media for Neisseria species growth.
Growth appears as Small, grayish-white to colorless
colonies.
BA:
Non-Selective differential media.
Important isolate is Group B streptococcus.
Growth appears as glistening gray-white colonies with a
narrow zone of β-hemolysis.
2-Wound swaps:
BA:
Non-Selective differential media.
Important isolate are;
-S. aureus → Big golden shiny colonies with wide zone of β-hemolysis.
-Enterococcus spp → Small transparent colonies with γ-hemolysis.
The rest are non-significant normal flora.
-All gram negative are significant in wound swaps.
3-Body fluid: Any growth is significant.

4-Throat swaps:
BA:
Non- Selective differential media.
Important isolate is Group A streptococcus.
Appears small colonies with small zone of β-hemolysis.
Urine bench:
A urine culture and analysis aim in looking for organisms that cause
UTI. This bench we do chemical analysis, pregnancy tests and
culturing.
The Set up will differ depending on the order requested;
 Urine analysis and culture
 Urine analysis only
 Urine culture only
The most important isolates:
- Enterobacteriaceae (Especially E.coli)
- Enterococcus faecalis
-Groub A and B streptococcus
-Staph aureus
-Pseudomonas spp.
-Candida spp.
Urine analysis:
DxU Iris Instrument:
An automated urine chemistry analysis that
delivers results in less than 2 minutes, it
composed of two parts;
-DxU 810c Iris for urine chemistry analysis.
-DxU 850m Iris for urine microscopic analysis.
Results on the software appears like this, The

right part is for the chemistry results and the left
part is for the macroscopic results.
The most significant indications for bacterial
growth are; ↑Nitrate, ↑WBC's and ↑Bacteria.
Classification Categories:
Category
RBC WBC WBCC SQEP NSE BACT
UNCX HYAL UNCC BYST SPRM MUCS
WBCC: White blood cell clumps, SQEP: Squamous epithelial cells, NSE: Non-
squamous epithelial cells, BACT: Bacteria, UNCX: Crystals, HYAL: Hyaline Casts
UNCC: Unclassified Casts, BYST: Yeast, SPRM: Sperm, MUCS: Mucous.
Quality control:
controls should be run at least once every 24 hours to verify and
monitor proper system operation.
DxU Microscopy 850m control:

-iQ Positive Control is used as an abnormal result.
-iQ Negative Control is used as a normal result.
-iQ Focus is used to check light level and focus the instrument.
iQ Positive Control and iQ Focus are suspensions of fixed human red
blood cells in a buffered.
DxU Chemical 810m control:

-IRISpec CA/CB/CC for monitoring of urine chemistry analytes.
Analytes: Bilirubin, Urobilinogen, Ketone, Ascorbic Acid, Glucose,
Protein, Blood, pH, Nitrite, Leukocytes, Specific Gravity.
Set up:
Culture depends on order request and sample condition.
1- UA order does not need culturing.
2- UA and Culter order → Culture on CLED agar.
NOTE: If one or all the significant indications are high, culture
on the purity plate as well.
3- Culture order→ Culture on CLED agar.
NOTE: If the urine sample is turbid, culture on the purity plate
as well.
Incubate the plates at 37 C° for 24h in O2 environment.
Reading the growth:
CLED:
A differential medium for isolating bacteria in urine and prevents swarming
of Proteus species.
Significant lactose fermenting species:
-S. aureus → Appears small and dry.
-E. coli → Appears large and dray(Klebsiella spp are mucoid).
-Enterococcus faecalis → Appears very small.
Significant non-lactose fermenting species:
- Pseudomonas → Appears large and mucoid with fruity smell, produces pigment.
- Groub B strep → Appears very small and colorless.
Yeast appears small, creamy and very shiny white colonies(Refer to mycology).
Columbia|MAC:
A differential medias for lactose fermentation and blood heamolysis.
MAC agar is selective for gram -ve bacteria.
Significant isolates:
-S. aureus → Ferment the lactose, give a wide zone of β- hemolysis.
-E.coli → Ferment the lactose, appears dry(Klebsiella spp are mucoid).
- enterococcus faecalis → Non-lactose fermenter, appears small and
transparent with γ-hemolysis.
TB lab
TB lab:
TB lab has high levels of isolation than other labs
considering working with highly contagious organisms such
as Mycobacterium species with a negative pressure
environment.
Safety precautions:
- Use a class-II biosafety cabinet to work on the samples
to avoid infection from aerosols.
- PPE must be worn all the time and MUST not leave the lab
with them to avoid spreading the pathogen.
Respirator fit testing:

A test done for the lab specialists that are exposed for infection.
It is important to ensure the respirator fits tightly to the face of the
wearer (user) and protects them from inhaling infectious aerosol
containing Mycobacterium tuberculosis.
Mycobacterium species
Mycobacterium species are a group of acid-fast, aerobic, slow-
growing bacteria.
The most important species is Mycobacterium tuberculosis, the
causative agent of tuberculosis.
Main characteristics:
 Cell wall is rich in lipid (Mycolic acid).
 Non-spore forming, non-motile and non-capsulated.
 Very slow growing.
 Dose not stain with gram stain.

 Tissue
 Body fluids
 Sputum
 Urine
 Blood
Samples are kept in a refrigerator in the bacteriology lab.
If the samples are not processed within 4 hours, we keep the
samples in the refrigerator at 8 C°.
NOTE: Upon receiving, MUST check the location from
where the sample came from, to make sure if its
contaminated with other bacteria or not.
It is important in culturing the sample to have a pure growth
of mycobacterium species only.
First step in processing the sample is;
Decontamination:
In order to get a pure and a viable culture, the specimens (from non-
sterile sites) must be treated with Sodium hydroxide solution(NaOH).
Aim in liquifying the sample and kill any other bacteria.
Add equal amount of both the sample and the NaOH solution, mix
and incubate the for 15min maximum.
Run a QC using a BA to check if there is any contaminations.
After decontamination, dilute the sample with 8-9ml of normal
saline. Then, centrifuge the sample for 10min at 3000RPM.
Discard the remining at the top and keep the sediments for culture.
Culture media:
There are 2 types of medias;
1- Solid media (Lowenstein Jensen medial).
o Add 3-4 drops of the sample.
→ Pyruvate
→ Glycerol
2- Broth media (Mycobacterium Growth Indicator tube)
o Add 500µl of the sample.
Make a slide smear for Acid Fast stain as well.

BD BACTEC™ MGIT™ 960 system

fully automated system for the rapid detection of
mycobacteria in clinical specimens. It is also used for the
antimicrobial susceptibility testing of mycobacteria.
Principle:
The MIGT media contains a fluorescence quenching based
oxygen sensor.
During the bacterial growth, the oxygen is consumed and replaced
with CO2. As a result, the fluorochrome inhibition by free oxygen
gradually diminishes, and its fluorescence in response to the
ultraviolet illuminations can be detected by the BACTEC's sensor.
PANTA™:
A soulution that contains 4 types of antibiotics. A kit come with the
MGIT™ 960 machine.
Aim in decontamination as well for other bacterias other than
mycobacterium.
NOTE: the MGIT™ 960 systen is very sensitiv, if the previous
decontamination process was not sufficient it will be detect
other bacteria and give Fals positive results.
We add 800µl of the PANTA solution in the MGIT tube.
Incupation period:
-Incupate the LJ medium at 37 C° for 8 weeks.
-Incupate the MIGT media at 37 C° for 6 weeks.
Staining:
-We stain the slide smear after the culture for the primary result.
There are 2 types of stain;
1-Primary stain (fluorecent stain)
→ Use a fluorecent microscope to observe.
2- Converb stain (Zihel-neelsen stain)
NOTE: We don’t do ZN stain unless we found the
mycobacterium in the primary stsin.
-We make a Gram stain as well to make sure if there is any
contamination with other bacteria.
Lab protocol: For confirmation, we run a PCR to confirm the result.

fluorecent stain:
Flurecent stain shows a high number of
organisms compared to ZN stain.
If there is a scanty amount of organism, it will
be difficult to see it in the ZN stain.
Organism will apear as Apple-green color.
Zihel-neelsen stain:
Since the mycobacterium containing waxy cell

wall(Mycolic acid), the heat will assist the stain
to enter the cell wall.
Acid fast bacteria will appear Red-pink .
Non-acid fast bacteria will appear Blue.
Sensitivity test:
Once we have a positive result for TB, we run a sensitivity test. It g is
valuable in the proper treatment of patients with tuberculosis
through a multiple drug regimen includes;
Streptomycin (STR), Isoniazid (INH), Rifampin (RIF) and Ethambutol
(EMB) called SIRE. Comes in a kit with their control.
Also, a new antibiotic called Pyrazinamide(PZA) come in a kit with its
control.
800µl of PANTA
100µl of SIRE
500µl of sample
GC STR INH RIF EMB
Incubate them in the BD BACTEC™ MGIT™ 960 system at for 14 days.
Hain test:
The GenoType® Mycobacterium CM12 assay is rapid and reliable
identification test of mycobacterial species other than tuberculosis.

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King Khalid University Hospital

King Saud University

Bacteriology lab include different benches; Stool bench, Urine

-Specimens should be opened in a Class-II biological

In all microbiology labs, the workflow for identification of

Modified Thayer-Martin agar (MTM II)

Other special medium

Todd Hewitt broth (TH)

Blood culture system

Test cards set up:

They are incubated and interpreted automatically and read

2- DxM MicroScan WalkAway ID/AST System:

ID section → Visual or instrumental read, biotype based on positive

Results of panels status:

The instrument have 8 towers, each has a 12 spots for panels.

The difference between VITEK®2 and MicroScan:

The antibiotic strip is then placed on to an inoculated agar

After incubation, bacterial growth becomes visible on the

Diagnostic methods used:

2-Coagulase test: Distinguish between staph species.

o Make a smear on a slide for gram stain.

Q-score (Quality Score):

NOTE: Patients under antibiotics treatment will have no organism

Q-score: points of average number of neutrophils + points of average

Squamous epithelial cells

The value Nill or Few Moderate ≥25

Q0 → Work only if the growth is predominant.

Q3 → Work up only 2 potential pathogens. A sample with a '+++'

A sample with no epithelial

Yeast (Not potential pathogen, sub-culture on SDA and refer to

Reading the growth:

Always start with the

o NO gram stain for stool, because it has a lot of microbiota.

If a C. diff test is ordered:

If a pathogenic E.coli (STEC O157 H7) is ordered:

STEC dose NOT ferment the sorbitol

B- Appearance of STEC that don’t ferments the

Reading the growth:

GN and Columbia agars are

GN→ isolation of gram –ve

2- Body fluid specimens:

o Make a smear on a slide for gram stain.

Bi plate, BA and MSA

o Make a smear on a slide for gram stain.

Ear and Eye swap Streps group B

Incubate the MAC, SDA and the Screening plate at 37 C° in O2

Reading the growth:

Growth in Swaps samples:

-All gram negative are significant in wound swaps.

3-Body fluid: Any growth is significant.

Results on the software appears like this, The

UNCX HYAL UNCC BYST SPRM MUCS

DxU Microscopy 850m control:

DxU Chemical 810m control:

Reading the growth:

Respirator fit testing:

Type of samples received:

First step in processing the sample is;

Make a slide smear for Acid Fast stain as well.

BD BACTEC™ MGIT™ 960 system