LESSON 1

LABORATORY EQUIPMENT

WHAT IS A LABORATORY?
 A room or place equipped for the performance of tests, experiments and investigative processes.
 Medical Laboratory – tests are done on clinical specimens in order to get information about the health of a
patient.

1. Microscope – an optical instrument consisting of a combination of lenses which magnifies the image of the
object seen through it.
 Microscopy – the examination of minute objects by means of a microscope
Micro – small Scope – to view
2. Incubator – a device used to grow and maintain microbiological cultures.
3. Autoclave – pressure chamber used to sterilize laboratory equipment and supplies by subjecting them to a
high temperature and high pressure.
4. Oven – a device for sterilization which uses dry heat
5. Laboratory Refrigerator – used to store stock cultures between sub culturing periods.
– maintains hydration of sterile media
– repository for thermolabile solutions, antibiotics and serums
6. Centrifuge – device which rotates at high speed and separates substances of different densities
7. Balance – used to measure object’s mass to a very high degree of accuracy
8. Hot plate/ Stir plate – used to heat and stir liquid substances for a faster and more even reaction
– the hot plate stirrer/ hot plate magnetic stirrer keeps liquids circulating as they
are heated
 Magnetic Stirring Bar – the stir bar is the magnetic bar which is immersed in the liquid to provide the
stirring action.
9. Water Bath – maintain water at constant temperature.
– used in the microbiological laboratory for incubations.
10. Logical Safety Cabinet – an enclosed, ventilated laboratory workspace for safety working with materials
contaminated with pathogens
11. Bunsen Burner – a common piece of laboratory equipment that produces a single open gas flame, which is
used for heating and sterilization.
12. Inoculating Loops and Needles – designed to pick up and transfer a small quantity of inoculum from a
donor culture to the growth medium of choice.
13. Anaerobic Jar/ GasPak – used to create an oxygen-free environment for the growth of anaerobic
microorganisms.
14. Glass Slides and Cover Slips
 Glass Slide – used to place specimens on to be observed under the microscope.
 Cover Slip – used to cover specimens on a microscope slide
15. Petri Dish – used to culture different types of cells, including bacteria and molds.
16. Graduated Cylinder – used to measure liquid volume.
– graduated in mL
17. Beakers – are vessels in which liquid is placed so it can be stirred, mixed or heated.
18. Erlenmeyer Flask – used to contain liquids and for mixing, heating, cooling, incubation, filtration, storage,
and other liquid-handling processes.
19. Funnel – used for guiding liquid or powder into a small opening
20. Test Tube/Rack
 Test tube – hold small amounts of substances, usually liquid, which are then manipulated in some
way, such as being placed over the flame of a Bunsen Burner.
 Tube Rack – hold multiple test tubes at the same time
 21. Wash Bottles – used to rinse various pieces of laboratory glassware, such as test tubes and round bottom
containers.
22. Filter Paper – special paper used to separate solids from liquids.

L ESSON 2 – REVIEW OF MICROSCOPE

Microscope History
 1665 – English physicist, Robert Hooke looked at a silver of cork through a microscope lens and noticed some
“pores” or “cells” in it.
 1674 – Anton Van Leeuwenhoek built a simple microscope with only one lens to examine blood, yeast, insects
and many other tiny objects. Leeuwenhoek was the first person to describe bacteria, and he invented new
methods for grinding and polishing microscope lenses that allowed for curvatures providing magnifications of
up to 270 diameters, the best available lenses at that time.
 18th Century – technical innovations improved microscopes, leading to microscopy becoming popular among
scientists. Lenses combining two types of glass reduced the “chromatic effect” the disturbing halos resulting
from differences in refraction of light.
 1830 – Joseph Jackson Lister reduces spherical aberration or the “chromatic effect” by showing that several
weak lenses used together at certain distances gave good magnification without blurring the image. This was
the prototype for the compound microscope.
 1872 – Ernst Abbe, then research director of the Zeiss Optical Works, wrote a mathematical formula called the
“Abbe Sine Condition”. His formula provided calculations that allowed for the maximum resolution in
microscopes possible.
 1903 – Richard Zsigmondy developed the ultramicroscope that could study objects below the wavelength of
light. He won the Nobel Prize in Chemistry in 1925.
 1931 – Ernst Ruska co-invented the electron microscope for which he won the Nobel Prize in Physics in 1986.
An electron microscope depends on electrons rather than light to view an object, electrons are speeded up in a
vacuum until their wavelength is extremely short, only one hundred-thousandth that of white light. Electron
microscopes make it possible to view objects as small as the diameter of an atom.
 1932 – Frits Zernike invented the phase-contrast microscope that allowed for the study of colorless and
transparent biological materials for which he won the Nobel Prize in Physics in 1953.
 1981 – Gerd Binnig and Heinrich Rohrer invented the scanning tunneling microscope that gives three-
dimensional images of objects down to the atomic level. Binnig and Rohrer won the Nobel Prize in Physics in
1986. The powerful scanning tunneling microscope is the strongest microscope to date.
TYPES OF MICROSCOPES
 Compound Microscope
 Compound microscopes are light illuminated. The image seen with this type of microscope is two
dimensional. This microscope is two dimensional. This microscope is the most commonly used. You
can view individual cells, even living ones. It has high magnification. However, it has a low
resolution.

 Dissection Microscope
 A dissection microscope is light illuminated. The image that appears is three dimensional. It is used
for dissection to get a better look at the larger specimen. You cannot see individual cells because it
has a low magnification. (Also called stereo microscope)
 Scanning Electron Microscope – SEM
 SEM use electron illumination. The image is seen in 3D. it has high magnification and high
resolution. The specimen is coated in gold and the electrons bounce off to give you and exterior
view of the specimen. The pictures are in black and white.
 Transmission Electron Microscope – TEM
  TEM is electron illuminated. This gives a 2D view. Thin slices of specimen are obtained. The electron
beams pass through this. It has high magnification and high resolution.
MICROSCOPE CARE
1. Always carry by the base and arm with both hands.
2. Never touch the lenses with your fingers.
3. Make sure all backpacks and materials are out of the aisles and off the tops of desks.
4. Plug your microscope in to the outlet.
MICROSCOPE PARTS
a) Ocular Lens – magnifies; where you look through to see the image of your specimen.
– they are usually 10x or 15x power. Our microscopes have an ocular lens power of 10x.
b) Arm – supports the tube and connects it to the base
c) Stage – the flat platform where you place your slides
d) Course Adjustment Knob – moves stage (or body tube) up and down
e) Fine Adjustment Knob – small, round knob on the side of the microscope used to fine-tune the focus of your
specimen after using the course adjustment knob.
f) Base – the bottom of the microscope, used for support
g) Body Tube – connects the eyepiece to the objective lenses
h) Revolving Nosepiece – the part that holds two or more objective lenses and can be rotated to easily change
power
i) Objective Lenses (adds to the magnification)
 Usually you will find 3 od 4 objective lenses on a microscope. They almost always consist of 4x, 10x,
40x and 100x powers.
 When coupled with a 10x (most common) eyepiece lens, we get total magnifications of 40x (4x
times 10x), 100x, 400x, and 1000x).
 The shortest lens is the lowest power, the longest ones is the lens with the greatest power. Lenses
are color coded.
 The high power objective lenses are retractable (i.e. 40XR). This means that if they hit a slide, the
end of the lens will push in (spring loaded) thereby protecting the lens and the slide.
j) Stage Clips – hold the slides in place. If your microscope has a mechanical stage, you will be able to move the
slide around by turning two knobs. One moves it left and right, the other moves it up and down.
k) Diaphragm
 Controls the amount of light going through the specimen. Many microscopes have a rotating disk
under the stage.
 This diaphragm has different sized holes and is used to vary the intensity and size of the cone of
light that is protected upward into the slide.
 There is no set rule regarding which setting to use for a particular power. Rather, the setting is a
function of the transparency of the specimen, the degree of contrast you desire and the particular
objective lens in use.
l) Light – makes the specimen easier to see
 Focusing Specimens
1) Always start with the scanning objective
2) Once you’ve focused on Scanning, switch to Low Power
3) Now switch to High Power
4) You can switch to the Oil Immersion but first put a drop of the Cargill’s oil in the center of the
lighted area.
5) Rotate until the OIO makes a click sound.
USING HIGH POWER
 Clean up! When you have finished for the day, wipe the 100x oil immersion objective carefully
with lens paper to remove all oil. Wipe oil from the slide thoroughly with a Kimwipe. Cleanse
 stage should any oil have spilled on it. Recap the immersion oil container securely, replace in
drawer.
LESSON 3 – ASEPTIC TECHNIQUE

Sterile Technique and Pure Culture Concept

Why?
1. To protect yourself from contact with bio hazards
2. To protect your sample from becoming contaminated
3. To protect others in the lab

ASEPTIC TECHNIQUE
a) Method of preventing unwanted microorganisms from gaining access
 The most commonly used device for moving bacteria is the inoculating loop.
 This is simply a piece of nichrome (an alloy of nickel and chromium) or platinum wire with a loop at one end
and a handle at the other.
 A similar instrument is the inoculating needle, essentially the same as the loop but with just a straight wire
 Inoculating loop or needle should be held comfortably , much as you would hold a pencil.

Sterilization of Instruments
1. Sterilize both instruments by holding the wire portion in a flame until they glow red. The instruments should
be allowed to cool in the air for 10-20 seconds before using them to avoid killing the inoculum.
2. In this way all contaminants on the wire are incinerated.
3. Do NOT blow on the instruments to cool them
4. Do NOT touch the instruments to agar to cool them
5. Do NOT lay the loop down once it is sterilized or it may again become contaminated.

The Procedure for Aseptic Transfer

1. Flame the loop.
2. Without setting the loop down, open the first culture tube and flame the mouth (why). Do not set the cap on
the bench. The cap should be held in the same hand as the loop.
3. Insert the loop into the culture medium, and then withdraw it.
4. Flame the mouth of the first culture tube again, and replace the cap.
5. Open the second culture tube and flame the mouth. Do not set the cap on the bench. The cap should be held
in the same hand as the loop.
6. Insert the loop into the second culture tube and spread the culture suspension (on the loop) inoculum
into/onto the second culture medium.
7. Flame the mouth of the second culture tube, and then replace the cap.
8. Flame the loop and set on the bench.
9. When in doubt about the sterility of an instrument or container, sterilize it.

FIVE (5) BASIC TECHNIQUES OF CULTURING

1. Inoculate
2. Incubate
3. Isolation
4. Examination/Inspection
5. Identification

PURE CULTURE CONCEPT

 Contaminants – other microorganisms present in the sample
  Isolated colonies – a population of millions of cells that are identical and are descendent from a single
founder cell
 Stock Culture – a culture that already contains cells.
 It is used a source of cells from which to inoculate new cultures.
 Culture Medium – rich/selective
 Growth inhibitors
 Liquid/solid
 Temperature
 Source of energy
 Sources of carbon, nitrogen, ....
 Aseptic Technique
 Sterilization of medium and equipment
 Proper handling
NECESSARY EQUIPMENT CULTURE MEDIUM
Inoculating Loop Agar Plate
Inoculating Needle Agar Deep Tube
Culture Tube Broth
Stock Culture (contain cells) Agar Slant
Bunsen Burner
Sterile Agar Plate
Test Tube Rack
PROCEDURE
1. With the loop, spread the inoculum back and forth across the upper ¼ of the plate, keeping the lines of
inoculation very close together (area 1 in figure below)
2. Isolated colonies are not expected in this area. Do not use strong pressure, which will break the surface of
the agar. Use the end of the loop, not its side when streaking. Dispose of the loop in the biohazard bucket on
the bench.
3. Turn plate approximately 90 degree. Streak the plate as indicated in the figure (area 2) across about ¼ of the
plate. Dispose of the loop.
4. Repeat step 2 one or two times more.
5. In area 3 and/or 4 single colonies should appear.
6. Label plates on the bottom and incubate inverted at 37 degrees Celsius.
Note. Lids on test tubes are loose. Always hold the glass test tube (not the lid) when carrying them.

FORMS OF CULTURE MEDIA

1. Broth tube – are tubes containing a liquid medium. A typical nutrient containing broth medium such as
Trypticase Soy Broth, nutrient broth. After incubation, growth (development of many cells from a few cells)
may be observed as one or a combination of three forms
a. Pellicle – a mass of organisms is floating on top of the broth
b. Turbid – the organisms appear as a general cloudiness throughout the broth
c. Sediment – a mass of organisms appears as a deposit at the bottom of the tube
2. Slant Tube – are tubes containing a nutrient medium plus a solidifying agent, agar-agar. The medium has
been allowed to solidify at an angle in order to get a flat inoculating surface.
3. Stab tubes (deeps) – are tubes of hardened agar medium which are inoculated by “stabbing” the inoculum
into the agar.
4. Agar plates – are sterile petri plates that are aseptically filled with a melted sterile agar medium and
allowed to solidify. Plates are much less confining that slants and stabs and are commonly used in the
culturing, separating, and counting of microorganisms.
LESSON 4 – CULTURE MEDIA AND CULTURE METHODS
HISTORY
 The original media used by Luis Pasteur – urine or meat broth
 Liquid medium – diffuse growth
 Solid medium – discrete colonies
 Colony – macroscopically visible collection of millions of bacteria originating from a single bacteria cell.
 Cooked cut potato by Robert Koch – earliest solid medium
 Gelatin – not satisfactory, liquefy at 24°C
 Agar
 Frau Hesse
 Used for preparing solid medium
 Obtained from seaweeds
 No nutritive value
 Not affected by the growth of the bacteria
 Melts at 98°C and sets at 42°C
 2% agar is employed in solid medium
TYPES OF CULTURE MEDIA
1. Based on their consistency
a) Solid medium
b) Liquid medium
c) Semi-solid medium
2. Based on the constituents/ ingredients
a) Simple medium
b) Complex medium
c) Synthetic or defined medium
d) Special media
 Enriched media
 Enrichment media
 Selective media
 Indicator media
 Differential media
 Sugar media
 Transport media
 Media for biochemical reactions
3. Based on Oxygen requirement
 Aerobic media
 Anaerobic media
I. ENRICHED MEDIA
 Substances like blood, serum, egg are added to the basal medium
 Used to grow bacteria that are exacting in their nutritional needs.
 E.g. Blood sugar, Chocolate agar
II. ENRICHMENT MEDIA
 Liquid media used to isolate pathogens from a mixed culture.
 Media is incorporated with inhibitory substances to suppress the unwanted organism.

 Examples:
 Selenite F Broth – for the isolation of Salmonella, Shigella
 Alkaline Peptone Water – for vibrio cholerae
 III. SELECTIVE MEDIA
 The inhibitory substance is added to a solid media
 Examples:
 Mc Conkey’s medium – for gram negative bacteria
 TCBS – for V.cholerae
 LJ medium – M.tuberculosis
 Wilson and Blair medium – S.typhi
 Potassium tellurite medium – Diphtheria bacilli
IV. INDICATOR MEDIA
 These media contain an indicator which changes its colour when a bacterium grows in them.
 Eg. Blood sugar, Mac Conkey’s medium, Christensen’s urease medium
V. DIFFERENTIAL MEDIA
 A media which has substance incorporated in it enabling it to distinguish between closely related
bacteria
 Examples: Mac Conkey’s medium
Peptone
Lactose
Agar
Neutral red
Taurocholate
 Distinguish between lactose fermenters and non-lactose fermenters
 Lactose fermenters – pink colonies
 Non-lactose fermenter – colourless colonies
VI. SUGAR MEDIA
 Media containing any fermentable substance
 E.g: glucose, arabinose, lactose, starch, etc.
 Media consists of 1% of the sugar in peptone water
 Contain a small tube (Durham’s tube) for the detection of gas by the bacteria
VII. TRANSPORT MEDIA
 Media used for transporting the samples
 Delicate organisms may not survive the time taken for transporting the specimen without a transport
media.
 Eg: Stuart’s medium – non nutrient soft agar gel containing a reducing agent
VIII. ANAEROBIC MEDIA
 These media are used to grow anaerobic organisms
 Eg: Robertson’s cooked meat medium, Thioglycolate medium
BIOCHEMICAL TEST & REACTIONS
 They provide additional information for the identification of the bacterium.
 The tests include:
- Oxidase test - Citrate utilization
- Triple sugar iron agar (TSI) - Urease test
- Indole test
 Oxidase Test
 Detects the presence of an enzyme “oxidase” produced by certain bacteria which will reduce the dye
– tetramethyl-p-phenylene diamine dihydrochloride
 Positive test is indicated by the development of a purple colour
 Oxidase positive – pseudomonas, vibrio, neisseriae
 Oxidase negative – Salmonella, Shigella
  Triple Sugar Iron Agar (TSI)
 It is a composite media used to study different properties of a bacterium – sugar fermentation, gas
production and H2S production
 In addition to peptone, yeast extract & agar, it contains 3 sugars – glucose, lactose, sucrose
 The Iron Salt – Ferric citrate indicates H2S production
 Phenol red is the indicator
 It is an orange red medium with a slant and a butt.
 pH of the medium – 7.4
TSI REACTIONS:
Yellow – Acid
Pink – Alkaline
 Yellow slant/ Yellow butt (A/A) – Lactose fermenters
 Pink slant/ Yellow butt (K/A) – Non lactose fermenters
 Pink slant/ no colour change (K/K) – Non fermenters
 Black colour – H2S production
 Gas bubbles or crack in the medium – gas production
 LF – E.coli, Klebsiella
 NLF – Salmonella, Shigella
 H2S – Proteus
 Indole Test
 Used to detect indole production by the organism.
 They produce indole from tryptophan present in peptone water.
 After overnight incubation, a few drops of indole reagent (Kovac’s reagent) is added
 Positive test is indicated by a pink ring
- Positive indole test – pink ring
- Negative indole test – yellow ring
 Indole positive – E.coli
 Indole negative – Klebsiella, Salmonella
 Citrate Utilization
 Done in Simmon’s Citrate medium
 To detect the ability of certain bacteria to utilize citrate as the sole source of carbon
 Contains Sodium citrate and bromothymol blue as the indicator
 If citrate is utilized, alkali is produced which turns the medium to blue
- Citrate positive – blue colour
- Citrate negative – green colour
 Positive – Klebsiella
 Negative – E.coli
 Urease Test
 Done in Christensen’s urease medium
 This test is used to detect organisms that produce urease
 Urease produced by the organisms split urea into ammonia and CO2
-Urease positive – pink colour
-Urease negative – yellow colour
 Positive – Proteus, Klebsiella
 Negative – E.coli, Salmonella
CULTURE METHODS
 Culture methods employed depend on the purpose for which they are intended
 The indications for culture are:
 – To isolate bacteria in pure culture
– To demonstrate their properties
– To obtain sufficient growth for the preparation of antigens and for other tests
– For bacteriophage & bacteriocin susceptibility
– To determine sensitivity to antibiotics
– To estimate viable counts
– Maintain stock cultures
Culture Methods include:
 Streak Culture
 Used for the isolation of bacteria in pure culture from clinical specimens
 Platinum wire or Nichrome wire is used
 One loopful of the specimen is transferred onto the surface of a well dried plate
 Spread over a small area at the periphery
 The inoculum is then distributed thinly over the plate by streaking it with a loop in a series of parallel
lines in different segments of the plate
 On incubation, separated colonies are obtained over the last series of streaks
 Lawn Culture
 Provides a uniform surface growth of the bacterium
 Uses
– For bacteriophage typing
– Antibiotic sensitivity testing
– In the preparation of bacterial antigens and vaccines
 Lawn cultures are prepared by flooding the surface of the plate with a liquid suspension of the
bacterium
 Stab Culture
 Prepared by puncturing a suitable medium
– Gelatin or glucose agar with a long, straight, charged wire
 Uses
– Demonstration of gelatin liquefaction
– Oxygen requirements of the bacterium under study
– Maintenance of stoke cultures
 Pour Plate Culture
 Agar medium is melted (15ml) and cooled to 45°C
 1 ml of the inoculum is added to the molten agar
 Mix well and pour to a sterile petri dish
 Allow it to set
 Incubate at 37°C, colonies will be distributed throughout the depth of the medium
 Uses
– Gives an estimate of the viable bacterial count in a suspension
– For the quantitative urine cultures
 Liquid Cultures
 Liquid cultures are inoculated by touching with a charged loop or by adding the inoculum with
pipettes or syringes
 Uses
– Blood culture
– Sterility tests
– Continuous culture methods
 Disadvantage
– It does not provide a pure culture from mixed inocula
ANAEROBIC CULTURE MEDIUM
 Anaerobic bacteria differ in their requirement and sensitivity to oxygen
 CI.tetani is a strict anaerobe – grows at an oxygen tension < 2 mm Hg
Methods:
a) Production of vacuum
 Incubate the cultures in a vacuum desiccator
b) Displacement of oxygen with other gases
 Displacement of oxygen with hydrogen, nitrogen, helium or CO2
 Eg: Candle jar
c) Chemical method
 Alkaline pyrogallol absorbs oxygen
McIntosh – Fikdes’ anaerobic jar
 Consists of a metal jar or glass jar with a metal lid which can be clamped air tight
 The lid has 2 tubes – gas inlet and gas outlet
 The lid has 2 terminals – connected to electrical supply
 Under the lid – small grooved porcelain spool, wrapped with a layer of palladinised asbestos
Gaspak
 Commercially available disposable envelope
 Contains chemicals which generate H2 and CO2 on addition of water
 Indicator is used – reduced methylene blue
– Colourless – anaerobically
– Blue colour – on exposure to oxygen
d) Biological method
 Absorption of oxygen by incubation with aerobic bacteria, germinating seeds or chopped vegetables
e) Reduction of medium
 By using reducing agents – 1% glucose, 0.1% Thioglycolate
LESSON 6 – STAINING PROCEDURES

STAINING

Differential Simple Special/Structural

Negative
Gram Acid-fast Endospore Flagella
(capsule)

PRE- STAINING PROCEDURE

Smear

Air-dry

Fix
(killm preserve,anchor)

SIMPLE STAINING

Pre-staining procedure

Basic dye is applied

(30-60 seconds)

Rinse and dry then view

GRAM STAINING
 Developed by Dr. Hans Christian Gram in 1884 which first demonstrated the general categories of
bacteria causing pneumonia
 Differentiates Gram-positive and Gram-negative bacteria

Pre-staining procedure

Crystal Violet (30-60 seconds)

Iodine (30-60 seconds)

Ethanol (30-60 seconds)

Safranin (30-60 seconds)