Chapter 1

Microscope
The microscope is used to produce magnified images of the minute organisms
which are examined by it.

I. The light microscope

The components of the light microscope can be classified into 4 systems:

A. Support system.

B. Optical (Magnification) system.

C. Illumination system.

D. Adjustment system.
A. Support system:

1. The foot.

2. The limb.

3. The stage.

B. Optical (magnification) system:

It consists of:
1- An external tube which bears at its lower end a revolving nose piece in which
several objective lenses are fitted.
2- An inner tube which carries the eye piece.

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C. Illumination system:
1- Source of light either a mirror for day light or an electrical source.

2- The condenser: It brings the rays of light to focus on the object.

3- The iris diaphragm: It lies just below the condenser and is used to control the
amount of light reaching the object.

D. The adjustment system:

It consists of:
1. The coarse adjustment screw: used first to achieve an approximate focus.
2. The fine adjustment screw: used to bring the object into a perfect focus.

Magnification:

• Definition: It is the power of the microscope to magnify objects and it is the

product of separate magnifications of the objective and the eye piece lenses.

• Magnification of the eye piece (× 10).

• Magnification of the objective lenses:

- Low power (× 10)

- High power (× 40)

- Oil immersion lens (× 80 - 100)

• Total magnification = Magnification of eye piece × Magnification of objective lens used.

Resolution:

Definition: The resolving power of the microscope is its capacity to reveal two closely
adjacent points as separate and distinct. The resolving power of the light
microscope is about 0.2 µm. The resolving power of the electron
microscope is one nm (nanometer).

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How to use the light microscope in bacteriological work?

In most bacteriological work, oil immersion lens is used.

* On using the oil immersion lens:

- A good light source is needed.

- Place a drop of cedar wood oil (Its refractive

index as that of glass) on the part to be
examined.

- Raise the condenser up as far as it will go.

- Open the iris diaphragm completely.

- Lower the (× 100) objective till it becomes in

contact with the oil using the coarse adjustment
(avoid pressing on the slide).

- Look through the eye piece; turn the fine

adjustment very slowly upwards till the image is
in focus. Light Microscope

II. The fluorescence microscope:

This microscope differs from light microscope in:

a. U.V. rays are used as a source of light instead of ordinary light.

b. Lenses made of quartz instead of glass.

[Link] fluorescent dyes are used for staining of bacteria or tissue, these fluorescent
dyes when exposed to U.V. rays will absorb it and convert it into visible rays and so
become luminous. This phenomenon is called fluorescence. Example of this
fluorescent dye is Auramine O used to stain T.B bacilli - yellow fluorescence.

d. The resolution power is 0.1 µm.

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Note: Fluorescein dyes can be conjugated to immunoglobulin and this is called
immunofluorescent technique and used for detection of antigens or antibodies.

Auramine-O staining of Acid-Fast Bacilli

Under Fluorescence Microscope
[Link]

III. The dark - ground microscope (oblique illumination):

It is used to visualize delicate

microorganisms (< 0.2 µm) as
spirochaetes which cannot be seen
by ordinary light microscope. A
special condenser is used which
allows oblique rays only to pass to
the object.

These oblique rays do not enter

the objective lens and do not reach
the eye unless they are scattered by
objects e.g. bacteria (spirochetes),
so the organisms appear brightly
illuminated in a dark background
(like stars in dark sky).

Dark - Ground Microscope

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IV. Electron microscope:

- The electron microscope is the only means by which most viruses can be seen. It is also
used to study the fine structure of viruses, bacteria or tissue cells.

- The source of illumination is an electron beam.

- Photographs are taken by camera placed below the screen.

- It has a resolving power of 1 nanometer (nm =1/1000 of a micron).

Electron Microscope
[Link]

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 Chapter 2

Morphology of Bacteria

• Size:
Most of pathogenic bacteria are measured in terms of microns (µ = 1/1000 of
millimeter). Bacteria range in size from 0.2 to 5 µm.

• Shape:
Bacteria are classified by shape into:

- Cocci: spherical in shape.

- Bacilli: rod shaped.

- Vibrio: comma shaped.

- Spirochaetes: spiral.
• Arrangement:
During cell division, the daughter cells may separate at once or may remain attached
to one another in pairs, clusters or chains.

Morphological study of bacteria:

To study the morphology of bacteria in the laboratory, microscopic examination should

be done. Fresh unstained preparations or wet films are examined for motility of bacteria.

Stained films are examined for study the morphology of organisms and their staining
reactions.

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Staining methods:

Proper examination of direct smears by different stains is very useful in several

situations:

1- Detection of organisms in direct smear may be sufficient for diagnosis as in:

- Tuberculosis. - Leprosy.

- Acute male gonorrhea. - Secondary case of cholera.

2- Information given by direct smear may be needed to start treatment before culture
results appear as in:

- Meningitis – Tuberculosis - Diphtheria-like organisms.

Types of staining:

I. Simple stains:

These stains show the morphology of the organism and the cellular elements in
exudates e.g. methylene blue stain in case of gonorrhea.

II. Differential stains:

a- Gram's stain:

It is the most important stain in microbiology. It separates most bacteria into two main
groups: The Gram-positive bacteria which are stained violet and Gram-negative bacteria
which are stained red.

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 Gram-Stained Films
[Link]

• Steps:

1. Prepare and fix smears.

2. Apply crystal violet for 1 minute.

3. Wash with tap water.

4. Apply iodine for 1 minute.

5. Add 95% (lipid solvent) e.g: ethyl alcohol rocking the slide from side to side. Pour
it off and re-apply till no violet color comes off the smear (Decolorization).

N.B: Ethyl alcohol extract the blue dye complex from the lipid rich thin-walled
gram –ve bacteria to a greater degree than from the lipid poor thick-walled gram
+ve bacteria.

6. Wash with water.

7. Counterstain with safranin or carbol fuchsin for 1 minute.

8. Wash with water, dry and examine under oil immersion lens.

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 Steps of Gram Stain
[Link]

b- Ziehl – Neelsen stain: (Acid fast stain)

It is used to stain Mycobacteria. This group of organisms possesses a thick waxy

envelope and requires special strong stain (carbol fuchsin and the application of heat for
their staining).

Once stained, they resist decolorization by sulphuric acid (acid fast). The pathogenic
members also resist decolorization by alcohol.

Ziehl – Neelsen Stained Film

[Link]

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� Procedure:
1. Prepare and fix smears.

2. Flood the slide with strong carbol fuchsin for 5-10 minutes. Heat intermittently, do
not allow the stain to boil or dry.

3. Wash with water.

4. Decolorize with acid alcohol (20% H2SO4 + 95% ethyl alcohol) until no more color
can be removed from the smear.

5. Wash thoroughly with water.

6. Counterstain with methylene blue for 2 min.

7. Wash with water. The film turns blue.

8. Dry and examine under oil immersion lens for acid fast rods (red against blue
background).

Steps of Ziehl – Neelsen Stain

[Link]

� Examples of acid-fast bacilli:

1- M. tuberculosis:
Resist 20% H2SO4 and 95% ethyl alcohol (acid fast and alcohol fast).

2- M. Leprae:
Resist 5 % H2SO4 and 95% ethyl alcohol (weak acid fast and alcohol fast).

3 - M. smegmatis: (Smegma bacilli are found in normal urethra in male and females)
Resist H2SO4 but not alcohol (acid fast and not alcohol fast).

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III. Special stains:

a. Capsule stain (India ink preparation):

i. Prepare a suspension of the organism with a drop of India ink on a slide and
cover with cover glass.
ii. Capsule will appear as clear area around the cell against dark background.

Capsulated Pneumococci by India Ink

[Link]

b. Staining of spores:

i. By Gram's stain, the body of the bacillus is deeply colored, whereas the spores
appear as unstained rounded or oval areas in the organism.

Spore Stained by Gram Spore Stained by Modified

Ziehl-Neelsen
[Link] [Link]

ii. By Modified Ziehl-Neelsen stain, in which decolorization is done with 0.5%

H2SO4, the spore appears pink and the body of the bacillus blue.

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 Chapter 3

Decontamination

DECONTAMINATION

CLEANING DISINFECTION STERILIZATION

Decontamination: is the use of physical or chemical means to remove, inactivate or

destroy pathogens rendering an object safe for handling. It includes:
• Cleaning,
• Disinfection, and
• Sterilization.
Cleaning: It is a process that removes foreign material (e.g. soil, organic material,
organisms) from the surface of an object. It should always precede disinfection or
sterilization.
Sterilization: It is the destruction of all forms of microorganisms including vegetative
forms and bacterial spores.

Disinfection: It is the destruction of most pathogenic microorganisms, but not

necessarily bacterial spores.

Choice of the level:

- The choice of the level of decontamination (cleaning, disinfection or sterilization)
depends on a number of factors:
• Type of material of object.

• Number and type of organisms involved.

• Risk of infection to patients or staff.

Spaulding’s classification: According to infection risk to the patient, equipment is
classified into three categories:

Low risk (non-critical): Items in contact with normal and intact skin e.g. blood pressure
cuff, stethoscopes.

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 o Cleaning followed by low or intermediate level disinfection is usually adequate.
Intermediate risk (semi-critical): equipment that does not penetrate the skin or enter
sterile areas of the body but is in contact with intact mucous membranes or non-intact
skin e.g. endoscopes, endotracheal tubes and thermometers.
o Cleaning followed by high level disinfection is usually adequate.
High risk (critical): items that penetrate sterile tissues, including body cavities and the
vascular system e.g. surgical, dental instruments and vascular catheters.
o Cleaning followed by sterilization is mandatory.
Cleaning:

Types: - Manual.

- Mechanical: washers and ultrasonic cleaners.

Cleaning agents: detergents

Methods of sterilization and disinfection

Sterilization and disinfection can be achieved either through physical or chemical

means:

DISINFECTION:

A. Physical means: heat and filtration

1- Heat:
• Boiling at 100°C:
- It is a high-level disinfection procedure.
Uses: HLD of scissors, knives, forceps, rubbers, and glass pipettes.

[Link]

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 • Steaming at 100°C (Koch's sterilizer):
N.B: Tyndallization: intermittent steaming 20 - 45 minutes for 3 successive days is the
method used for sugar media which decompose at higher temperature. In this process
all non-sporing organisms are killed and when the spores left in the medium at room
temperature will germinate and on the second exposure these vegetative forms will be
killed and so on with the third exposure. It is not considered to be a method of
sterilization as some spores may survive and later germinate and multiply.

• Moist heat below 100oC:

1. Pasteurization

2. Vaccine bath

3. Inspissation: exposure of the culture medium to humid heat at 75°C for

two hours on 3 successive days. This method used for serum-based
media e.g. Loffler's serum or egg based media e.g. LJ and Dorset egg
medium.

2- Filtration:

This a mechanical method used for fluids which are destroyed by any type of heat
e.g. serum, plasma, fluid culture media, vaccines, antibiotics and hormones. They are of
very minute pores, that hold back any bacteria or rickettsia but not viruses. Filtration can
be done by:

1. Seitz filter: It consists of asbestos disc. The apparatus with the asbestos
disc is sterilized in the autoclave before use.

2. Millipore filters: These are synthetic membranes made from cellulose

acetate. They can be used to study sizes of viruses.

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 Seitz Filter

Millipore Filter
[Link]

[Link] methods: by either disinfectants or antiseptics:

Disinfectant:

- Kills micro-organisms (bactericidal).

- Used to disinfect inanimate objects only as it is a potent toxic substance.
e.g. phenolic and chlorine compounds.
Antiseptic:
- Inhibits growth of micro-organisms (bacteriostatic).
- Suitable for superficial application to living tissues e.g. wounds as it is a
less toxic substance. e.g., iodophore compounds.

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a. Low level disinfectants (LLD):

QUATs

b. Intermediate level disinfectants (ILD):

Ethanol, Betadine & Chlorhexidine (skin antisep�cs)

chlorine (disinfectant)

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 c. High level disinfectants (HLD):

Hydrogen Peroxide 7.5% for 30 min for HLD

Hydrogen Peroxide 3-6% for wound an�sepsis & mouth gargle.
[Link]

Glutaraldehyde (Cidex) 2% for 20-90 min.

Ortho-Phthaldehyde (OPA) 0.5 % for 12 min.
[Link]

N.B. Some chemicals can be used as both disinfectant and antiseptic such as alcohol.

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Air disinfection:

To decrease airborne contamination level in high-risk areas of healthcare facilities e.g.:

Operating rooms, laboratories and pharmaceutical settings.

Methods that can be used for air disinfection:

1- Air filtration: using HEPA (High Efficiency Particle Arresters) filters.

HEPA Filter HEPA Filter in safety cabinet

2- Ultra-violet ray: It should be used in combination with HEPA filters.

Ultraviolet lamps in opera�ng room

[Link]

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 STERILIZATION: Can be also achieved by physical or chemical means:
I. Physical means: including
a) Heat.
b) Radiation.
A. Heat:
1- Steaming under pressure (The autoclave).
2- Dry heat (hot air oven)
1- The autoclave:
Principle: When the pressure increases above atmospheric, the boiling point also
increases. This is the same principle of both the pressure cooker.

Uses: sterilization of surgical instruments, bed linen, surgical dressing, gowns, cotton,
gauze, and for any culture media not destroyed by heat.

Temperature and timing of use:

• 121˚C for more than 15 minutes.
• 134˚C for 3 minutes.

Simple Autoclave & Items Sterilized by It

[Link]

Steam sterilization monitoring (sterilization assurance indicators):

1) Mechanical methods: through recording cycle time, temperature and

pressure.
2) Chemical methods: that will change its color or shape according to time and
exposure to temperature. They may be:
- External chemical indicators: indicates exposure of the package to

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 temperature.
- Internal chemical indicators: indicates penetration of temperature inside
the package.
3) Biological methods (Spore indicator): Bacillus stearothermophilus is the
standard spore-bearer used. It is killed at 121°C for 12 minutes. A small strip
of filter paper soaked in suspension of this organism is placed in the middle
of the autoclave and after sterilization is completed the organism is tested for
viability.

- External chemical indicators

[Link]

- Internal chemical indicators

[Link]

- Biological methods (Spore indicator)

[Link]

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2- Hot air oven:
Uses: sterilization of glassware such as test tubes, petri dishes, flasks, pipettes and all
glass syringes.
Temperature and timing of use:170 - 180°C for 1-2 hours.

B. Radiation (also called Cold sterilization)

Ionizing radiation: e.g. Gamma rays from cobalt 60.
Uses: sterilize disposable plastic syringe, catheters, gloves, and sterile disposable
swabs which are used for taking specimens from throat, conjunctiva, vagina or rectum.

~ 24 ~
 Items Sterilized by Ionizing Radiation

Chemical sterilants: Mainly used for heat labile critical items (e.g. endoscopes, some
dental instruments)
• 2% GLUTRALDEHYDE for 10 hours.
• 7.5% HP for 6 hours.

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 Hydrogen Peroxide 7.5% for 6 hours
[Link]

Glutaraldehyde (Cidex) 2% for 10 hours

[Link]

N.B. Some chemicals can be used as LLD, ILD, HLD or chemical sterilizers according
to their concentration and/or time of exposure.
Low temperature sterilization: used for heat or moisture sensitive items e.g. fiber-
optics, plastic, rubber, cat-gut, silk, fur and heart - lung machines.

- Ethylene oxide gas (ETO): used in low temperature of 35-65°C. Exposure

time for 1-6 hours. Bacillus subtilis can be used as its biological indicator.
- Plasma gas (hydrogen peroxide): used in low temperature of 35-45°C.
Exposure time for 75 minutes.

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 Ethylene Oxide Sterilizer Plasma Gas Sterilizer
[Link] [Link]

Applications of different methods of sterilization:

Item Methods of sterilization

Most of culture media Autoclave

TCBS, selenite F broth Steaming at 100°C
Tyndallization (special method of
Sugar medium
steaming)
Loffler’s serum, Dorset egg, LJ Inspissation
Hormones, antibiotics, serum Filtration
Bacteriological loop Red heat
Mouth of tubes and flasks Flaming
Glassware ([Link] tubes, flasks) Hot air oven
Rubber gloves, catheters & Plastic
Gamma irradiation
syringes

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 Chapter 4

Culture Media
Cultivation of bacteria is a process in which artificial media with known nutritive
components is used to allow bacterial growth and multiplication in vitro. Bacteria are
growing in the laboratory for several purposes:

a) To study their properties.

b) To isolate them from pathological specimens e.g. sputum, urine, pus or blood for
diagnosis, purification, identification and determination of their antibiotic
sensitivity.
c) To prepare antigens, toxins, vaccines or other products.

Culture media:
According to the consistency, there are two broad groups of media, liquid and solid:

I. Liquid media:

- Examples of liquid media are: peptone water and nutrient broth.

- The growth of bacteria in liquid media appears as turbidity of the clear fluid or
in the form of sediment or surface pellicle.
- They are used mainly for enrichment of the bacteria present in the
pathological samples and for biochemical testing.
- They could not be used in:
a- Identification of bacteria because if there is more than one type of bacteria
in the pathological sample.
b- They don't permit colony count.

II. Solid media:

- Examples of solid media are nutrient agar, blood agar and MacConkey’s
agar.
- The growth of bacteria on solid media appears as colonies (large groups of
bacteria).

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 - They are used for isolation and identification of bacteria, colony count as well
as for testing antimicrobial sensitivity.
Types: It can be classified into:

A. Ordinary (basal) media.

B. Enriched media.

C. Differential media.

D. Anaerobic culture media.

E. Blood culture media.

F. Transport media.

Note: Before culturing the patient specimen on a culture medium, you must stop
the antibiotic (as possible) for 48 – 72 h.

A. Ordinary (basal) media:

They contain the essential growth requirements and are sterilized in autoclave.

• It is used for:
i. Growing organisms that do not have special growth requirements (non –
fastidious).
ii. Base for other media.

• It includes:
1. Nutrient broth.
2. Peptone water.
3. Nutrient agar (plate or slope to increase the surface area): It is composed of
nutrient broth + 2 % agar.

Note: Soft agar is composed of nutrient broth+ 0.7% agar.

Methods of culture: Stabbing or inoculation.

Uses: - Stock culture. - Testing organism’s motility.

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 Soft Agar Peptone Water Nutrient Broth
[Link] [Link] [Link]

Nutrient Agar Plate Nutrient Agar Slope

[Link] [Link]

B. Enriched media:

They contain in addition to the essential growth requirements, highly nutritive substances
such as blood, serum or egg for growth of fastidious organisms such as Neisseria.

It includes:
1. Blood agar plate:

- Composition: Nutrient agar + 5 - 10% sheep, horse, ox or expired human blood.

- Sterilization: Nutrient agar by autoclave + sterile blood is added to it when its
temperature reaches 55°C, then it is poured in plates.
- Uses:

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 i. Cultivation of fastidious organisms as Streptococci, Pneumococci, Neisseria
and Haemophilus.

ii. Test hemolytic activity of some organisms:

o ß- hemolytic organisms; colonies surrounded by clear zone of complete

hemolysis as Streptococcus pyogenes, Staphylococcus aureus and
Clostridium tetani (They produce hemolysin toxin).

o α- hemolytic organisms; colonies surrounded by greenish zone of incomplete

hemolysis as Streptococcus pneumoniae & viridans Streptococci (They don't
produce hemolysin toxin).

o Non hemolytic as Staphylococcus epidermidis.

Blood Agar Hemolysis on Blood Agar

[Link] [Link]

2. Chocolate blood agar plate:

- Composition: As blood agar but after addition of blood, we increase the
temperature of water bath to 80°C for 10 min.
- Uses: Cultivation of Neisseria and Haemophilus.

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 Chocolate Agar
[Link]

3. Loffler's serum slope:

- Sterilization: Inspissation (70-80°C / 2h / 3Days).
- Uses: Cultivation of Corynebacterium diphtheria.

Loffler's Serum Slope

[Link]

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 4. Dorset’s egg medium (Slope, screw capped to inhibit dryness)
- Sterilization: Inspissation.
- Uses: Cultivation of T.B from sterile sample e.g. C.S.F.

Dorset’s Egg Medium

C. Differential media:

These media help to differentiate certain bacteria from others.

It includes Selective, enrichment and indicator media.

� Selective media: They contain inhibitory substances that inhibit organisms not desired
to grow while the organism under study will grow (selective agents).

1. MacConkey’s agar plate:

- Composition: Peptone water + agar + bile salts (inhibit the growth of non-intestinal
bacteria) + lactose + neutral red (indicator that gives pink in acidic pH &pale
yellow in alkaline pH)
- Sterilization: Autoclave.
- Uses: i. Cultivation of enteric bacteria.
ii. Differentiation between enteric bacteria:
o Lactose fermenters give pink colonies as [Link].
o Non lactose fermenters give pale yellow colonies as Salmonella.

~ 33 ~
 MacConkey’s agar
[Link]

2. Lowenstein Jensen media (L.J) (slope, screw capped):

- Composition: Whole beaten eggs + mineral salt + malachite green (selective
agent). In addition of glycerol for human type & pyruvate for bovine type for
enhancement.
- Sterilization: Inspissation.
- Use: Cultivation of tubercle bacilli from contaminated specimen as sputum,
urine and stool.

Lowenstein Jensen Media

3. T.C.B.S (Thiosulphate Citrate Bile salt Sucrose):

- Composition: Nutrient agar + Na thiosulphate, Na citrate and bile salt (selective
agents) + sucrose + bromothymol blue (indicator that becomes yellow in acidic pH
&blue in alkaline pH)).
- Sterilization: Steaming at 100°C for 30-60 min.

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 - Uses:
i. Selective for isolation of Vibrio species.
ii. Differentiation between different Vibrio species: Those ferment sucrose give
yellow colonies as V. cholera.

T.C.B.S
[Link]

4. Mannitol salt agar:

- Composition: Agar + mannitol + NaCl (10-15%) that inhibit any organism other than
Staphylococci + phenol red indicator that gives yellow color in acidic pH.
- Sterilization: Autoclave.
- Uses: Isolation of Staphylococci (Staph. aureus will be surrounded by yellow
haloes).

Mannitol Salt Agar

~ 35 ~
� Enrichment media:
1. Selenite F broth:
- Sterilization: Steaming at 100°C.
- Uses: Enrichment media for isolation of Salmonella and Shigella from
stool (inhibit coliform bacilli).

2. Tetrathionate broth:
- Sterilization: Autoclave.
- Uses: as Selenite F broth.

Selenite F Broth Tetrathionate Broth

� Indicator media:
1. Sugar media:
- Composition: Peptone water +1% tested sugar as glucose, sucrose, lactose or
maltose + Andrade’s indicator (pH indicator which becomes red in acidic pH
and pale yellow in alkaline pH) + small, inverted Durham's tube immersed in
the media for gas collection in fermenting organisms with gas production.
- Sterilization: Tyndallization (Steaming / 30 min / 3 days).
- Uses: Test the fermentative activity of bacteria.

~ 36 ~
 Sugar Media Sugar Media Sugar Media
(Fermentation with (Fermentation with (No
acid and gas) acid only) fermentation)

2. Triple sugar iron agar (slope):

- Composition: Nutrient broth + soft agar to crack on gas production + 3 sugars: 0.1%
glucose, 1% lactose, 1% sucrose + phenol red (indicator which becomes yellow
in acidic pH and pink in alkaline pH) + ferrous sulfate (for H2S detection).
- Sterilization: Autoclave.
- Uses: Differentiation of bacteria;
o Bacteria that ferment glucose only, will release a small amount of acid and
give a yellow butt and a red slant e.g. Shigella.
o Bacteria that ferment glucose only with H2S production will give a black butt
and a red slant e.g. Salmonella and Proteus (If no gas � S. typhi, but if
with gas � S. paratyphi B or Proteus).
o Bacteria that ferment glucose and lactose and/or sucrose will release a large
amount of acid and gas and give a yellow slant and butt e.g. [Link] and
Klebsiella.
o Bacteria with no carbohydrate fermenting activity will give a red slant and butt
e.g. Pseudomonas aeruginosa.

~ 37 ~
 Triple Sugar Iron Agar
[Link]

D. Anaerobic culture medium:

Principle: Anaerobic conditions can be achieved by:

• Addition of anaerobic agent to the media e.g. Robertson’s cooked meat broth.
• Removing of oxygen from the surrounding atmosphere e.g. anaerobic jar.

2. Robertsons's cooked meat broth:

- Composition: Nutrient broth + minced cooked meat.
Mincing of meat leads to the release of reducing substances such as
haematin and glutathionine which reduce O2 from the environment.
- Sterilization: Autoclave
- Uses: Cultivation of anaerobic organisms as Clostridium, Lactobacilus Bifidus and
Bacteroids.

~ 38 ~
 Robertsons's Cooked Meat Broth
[Link]

3. Anaerobic Gaspak Jar:

- A special plastic jar with a tightly fitting lid is used. Suspended from the lid is a
cold catalyst, which consists of aluminum pellets coated with palladium.
- In this system, a special Gaspak envelope commercially prepared is used. It will
release hydrogen and CO2 under pressure.
- The presence of the cold catalyst in the jar allows the hydrogen released to combine
with the remaining oxygen in the jar to give strictly anaerobic condition.
- An anaerobic indicator is used e.g. methylene blue which is colorless and
changes blue in the presence of O2. So that, a paper soaked with methylene blue
placed in an aluminum packet, is put in the jar to check the activity of the catalyst.
- Used for culture of strict anaerobic organisms as Clostridium, Bacteroides &
Peptostreptococci.

Note: To cultivate in an atmosphere with excess CO2 (e.g. Brucella abortus and
pathogenic Neisseria which requires 5-10% CO2), anaerobic jar can be used. But instead
of Gaspak and catalyst, we lighten a candle in the jar just before putting on the lid
(candle jar).

~ 39 ~
 Anaerobic Gaspak Jar
[Link]

E. Blood culture media:

Direct plating of the blood specimen on culture media for isolation of bacteria causing
bacteremia or septicemia is not recommended because:
1. Organisms are circulating in a few numbers in the blood stream.
2. Blood may contain antibodies or any inhibitory substance that will interfere
with bacterial growth.

So for culturing of blood specimens, blood culture bottles containing 50-100 ml of

sulphonated broth are needed (Na sulphonate is an anti-coagulant).

The big volume of broth has the following advantages:

i. It dilutes out antibodies or natural antibacterial substances in the serum.
ii. It provides good nutrient condition for multiplication of the organism.
iii. If the patient is taking antibiotics, antagonists to that antibiotic can be
added to the fluid medium e.g. penicillinase is added if the patient is taking
penicillin.

There are several blood culture systems are commercially available:

1. Monophasic blood culture medium:
- In which, the bottle contains only sulphonated broth (any additional nutrient
is added according to the growth requirements of the isolated organism).

~ 40 ~
 - For anaerobic organisms, sodium thioglycolate is added as a reducing
agent.
- Subculture is done from this culture every 48h on suitable solid media and
blood culture is considered negative only if no growth appears after 10
days.

2. Diphasic blood culture medium (Castaneda):

- That combines a broth medium (liquid phase) with a solid agar medium
(solid phase) in the same bottle.
- It has the advantage of decreasing the incidence of contamination present in
monophasic type from repeated subculture, as the organism present appear
on the agar sheet in the same bottles.

3. Automated blood culture systems: That monitor early signs of

bacterial growth are also available.

Precautions to obtain an optimum blood culture result:

• Use a sterile syringe after through sterilization of the puncture area with 70%
ethanol and betadine to avoid contamination of the specimen with skin commensal
organisms.

• Blood should be withdrawn from the patient when the fever is at its highest peak.

• Blood is collected before the start of antibiotic therapy. If an antibiotic has already
been taken, the sample should be taken just before the next antibiotic dose,
otherwise antagonist should be added to the broth.

• 3 specimens are taken from 3 different sites.

Indications of blood culture:

1- Subacute bacterial endocarditis.
2- Enteric fever.
3- Brucellosis.
4- Meningitis.
5- Puerperal sepsis.

~ 41 ~
 6- Osteomyelitis.
7- Pneumonia.

Monophasic Blood Culture Diphasic Blood Culture

[Link] [Link]

F. Transport media:

Used for preservation of delicate pathogens during transport to the laboratory e.g.
Stuart’s media used for N. gonorrhea.

Stuart’s Transport Media

[Link]

~ 42 ~
Different Ways of Inoculating Culture Media
[Link]/doc/18059761/Monica-Cheesbrough-District-
Laboratory-Practice-in-Tropical-Countries-Part2

~ 43 ~
 Chapter 5

Antibiotic Sensitivity Tests

This can be determined by three in vitro tests:

1- Disc diffusion method:

This is the most used method for determining antibiotic sensitivity.

Steps:
1) Using a sterile cotton swab dipped in the test culture (108 organisms /ml),
streak evenly over the prepared Mueller-Hinton agar plates.
2) Allow the plates to dry, then using a fine pointed forceps; distribute the
filter paper antibiotic discs (each disc contains certain concentration of one
antibiotic) onto the plates.
3) After overnight incubation at 37�C, zones of growth inhibition will appear
around those antibiotic discs to which the organism being tested is
susceptible.
4) Those zone diameters must be carefully measured using metric rulers
viewing from the back of the Petri dish.
5) According to the diameter of the inhibition zone, as read from standard
charts, for the different antibiotics, it can be determined if the organism is
highly or moderately sensitive or resistant to the different antibiotics.

Antibiotic Sensitivity Plate

[Link]

~ 44 ~
 Zone Diameter Interpretative Standards For Staphylococcus
aureus
[Link]

2- Tube dilution method:

The use of this method is limited to certain situations as:

a. If there is no response to antibiotic treatment although adequate amount is

applied.
b. Relapses during antimicrobial treatment.
c. Immunosuppression.

Steps:
1) Two-fold dilution of the sensitive antibiotic is done in a suitable nutrient
fluid medium.
2) A constant volume of a 24 h broth culture of the isolated organism (105
organisms/ ml) is added to each tube.
3) Control tube containing no antibiotic is included in each test.
4) After incubation at 37°C for 24 h, the tubes are examined for turbidity.
5) Minimal inhibitory concentration (MIC): The tube with the highest dilution
(lowest concentration) showing no visible turbidity (it is the least
concentration of the drug that inhibits bacterial growth).
6) Subcultures from MIC tube on solid media without antibiotic can be made
to know if the drug is bactericidal or bacteriostatic. If no growth occurs,
so the organism is dead, and this concentration is known as the minimal
bactericidal concentration.

~ 45 ~
 Note: Minimal bactericidal concentration (MBC): The tube with the highest
dilution that fails to yield growth on the subculture plate without
antibiotic (it is the least concentration of the drug that kills bacteria).

Tube Dilution Antimicrobial Susceptibility Testing

Epsilometer or “E” Test:

• It is a new technique for direct determination of the MIC.
• A gradually increasing concentration of the antibiotic is fixed along a
rectangular plastic test strip.
• The strip is applied to the surface of an inoculated agar plate.
• After overnight incubation, a teardrop-shaped inhibition zone is seen.
• The zone edge intersects the graded test strip at the MIC of the antibiotic.

E-Test
~ 46 ~
 Chapter 6
Identification of Isolated Bacteria

� The main methods of identification are:

1- Microscopic examination:
− Unstained smear (wet smear): to demonstrate the motility of the
organism.
− Stained smear: to demonstrate the staining reaction of the organism,
their morphology and their arrangement. Gram stain is most commonly
used.

2- Cultural appearance:
− The colonial morphology: shape, size and the color of the colony.
− Pigment production: endopigment e.g. golden yellow pigment of
Staphylococcus aureus or exopigment e.g. Pseudomonas aeruginosa.
− Presence of hemolysis: e.g. β-hemolysis of Staphylococcus aureus, α-
hemolysis of Viridans streptococci.

3- Biochemical reactions

4- Slide agglutination: can be used in identifying a given bacterium by using

specific antiserum.

5- Molecular methods for detection and identification of microorganisms:

All organisms contain in their genome some unique sequences of DNA or
RNA that distinguish them from other organisms.

Biochemical reactions (BR)

Bacterial species differ in their power to ferment carbohydrates or to split

proteins and also in the end product of this break down. This criterion is of great
benefit in identifying and in classifying the different bacterial species.
The saccharolytic activity:

It is done by growing the organism on a special sugar medium containing the

test sugar and indicator, which changes its color when pH becomes acidic, and an
inverted Durham's tube, dipped completely in the sugar media.

If gas is produced this will be evident by the presence of a gas bubble at the
top of the Durham's tube. Thus, we can say this organism is fermenting or not, any of
the tested sugar with the production of acid only or acid and gas, e.g. E. coli ferment
glucose with the production of acid + gas. (see chapter 4)

~ 47 ~
 The action of bacteria on milk is also differentiating, as some bacteria turn it
acidic and clotted while some turn it alkaline.

The proteolytic Activity:

It can be measured by the ability of the organism to liquefy protein gels as

gelatin and solidified serum as well as production of indole, H 2S or ammonia from
peptone.

Indole production: It is tested by growing the organism on peptone water for

1-2 days. Any indole produced is extracted first by xylol to float to the top. Then
addition of Kovac’s reagent → pink color i.e., positive case.

H2S production: Tested by holding a lead acetate-soaked filter paper at the

mouth of the tube of peptone water medium. Blackening of the paper means positive
result i.e., H2S production.

~ 48 ~
Other special tests may be used for differentiation of the Gram-negative intestinal
organisms as citrate, urease, Voges-Proskauer's and methyl red tests.

Citrate test:

Test the ability of organism to use citrate as the only carbon

source. Prepare slopes of the medium in bijou bottles. Using a sterile
straight wire, first streak the slope with a saline suspension of the test
organism and then stab the butt. If a bright blue color appears in the
medium, the test is positive.

Urease test:

The test organism is cultured in a medium which contains urea and the
indicator phenol red. When the strain is urease producing, the enzyme will break
down the urea (by hydrolysis) to give ammonia and carbon dioxide. With the release
of ammonia, the medium becomes alkaline as shown by a change in color of the
indicator to pink red.

~ 49 ~
 Voges Proskauer Test (V.P): the microorganism is grown on glucose
phosphate peptone for 2 days then add one ml of 10% caustic potash (KOH). If the
reaction gives pink color, the test is positive.

Methyl Red test (M.R): this test depends on the final acidity that occurred in
the glucose phosphate so on adding methyl red indicator if the test is positive, it will
give red color but if it is negative, it will give yellow color.

Oxidase test: The oxidase test is used to assist in the identification of

Pseudomonas, Neisseria, Vibrio and Brucella, all of which produce the enzyme
cytochrome oxidase. A piece of filter paper is soaked with a few drops of oxidase
reagent. A colony of the test organism is then smeared on the filter paper.
Alternatively, an oxidase reagent strip can be used. When the organism is oxidase-
producing, the phenylenediamine in the reagent will be oxidized to a deep purple
color. Occasionally the test is performed by flooding the culture plate with oxidase
reagent.

~ 50 ~
Bile solubility test:

This helps to differentiate S. pneumoniae, which is soluble in bile and bile

salts, from other alpha hemolytic streptococci (viridans streptococci) which are
insoluble. A heavy inoculum of the test organism is emulsified in physiological saline
and the bile salt sodium deoxycholate is added. This dissolves S. pneumoniae as
shown by a clearing of the turbidity within 10–15 minutes. Viridans and other
streptococci are not dissolved and therefore there is no clearing of the turbidity.

Catalase test:

This test is used to differentiate those

bacteria that produce the enzyme catalase, such
as staphylococci, from non-catalase producing
bacteria such as streptococci. A drop of hydrogen
peroxide solution is placed on a slide; a small
amount of the bacterial growth is emulsified in the
solution. The formation of bubbles (due to release
of O2) indicates a positive test.

2 H2O2 � 2 H2O + O2

~ 51 ~
Coagulase test: This test is used to identify S. aureus which produces the enzyme
coagulase (cell bound or free) which coagulates plasma, while other Staphylococci
are coagulase negative.

It can be tested by two methods:

1. Slide method: detects coagulase bound to the organism

• It is done on a microscopic slide using a drop of undiluted plasma on one side
and a drop of sterile saline on the other side as a control, while part of Staph
colonies is emulsified with each side separately.
• Clumping of Staph together means a positive test, while the saline side must
not show any clumping.
2. Tube method: detects free coagulase.
• Diluted citrated rabbit or human plasma is mixed with an equal volume of
broth culture of Staph in a test tube and incubated at 37ºC.
• A tube containing plasma and sterile broth is used as a control.
• If clot is formed in the test tube after 1-6 hours, the test is positive.

Slide coagulase test Tube coagulase test

~ 52 ~
 Chapter 7

Laboratory Diagnosis of Viral Infections

I. Detection of :

A. Virus Electron Microscope, Light Microscope (inclusion bodies).

B. Viral Ag Immunofluorescence

C. Viral nucleic acid PCR

II. Isolation: Tissue culture or egg inoculation.

III. Serologically: Detection of antibodies by:

1. ELISA.

2. IF.

3. Hemagglutination inhibition test

4. Neutralization test
a) Detection of the virus particles in patient specimen: by

1. Electron microscope:

Adenovirus under Electron Microscope

[Link]

~ 53 ~
 2. Detection of inclusion bodies:

Intracytoplasmic Inclusion Bodies

b) Detection of viral antigen in patient specimen:

Immunofluorescence:

Immunofluorescence Microscopy of Rabies Virus

[Link]

c) Detection of viral nucleic acid in patient specimen: by PCR.

~ 54 ~
 ➢ Viral isolation: by
a) Tissue culture

Tissue Culture Bottles

[Link]

Types of (TC) according to cells used:

1. 1ry TC: formed from organs: e.g., monkey kidney.
2. 2ry TC (Diploid TC): from embryonic fibroblasts.
3. Continuous TC: From malignant cells.

Preparation of Tissue Culture from Primary Cells

[Link]

b) Egg Embryo: Different cavities of the embryo could be used for inoculation e.g.
Amniotic sac: for influenza virus.

~ 55 ~
Diagnosis of viral infection in TC:

a) Cytopathogenic effect (CPE): These are the microscopic changes in the

cells such as: lysis, loss of intercellular bridge (giant cells or syncytium).
Then, we do neutralization test to determine the virus

Normal Monolayer of Tissue Culture Cells

(Continuous Sheet of Normal Cells)

Inoculated Tissue Culture Cell Line Showing:

cytopathic effect in the form of areas devoid of cells due
to their lysis (days after inoculation)

~ 56 ~
 Multinucleated Giant Cell

b) Neutralization test: absence of CPE of the virus when cultured after

mixing with specific antibodies.

Neutralization Test in TC
c) Immunofluorescent staining (fluorescence-labeled antibodies against
viral specific Ag).

Immunofluorescence Microscopy of West Nile Virus

Infected Vero Cells
[Link]

~ 57 ~
➢ Serologically: Detection of antibodies by:

1. ELISA.

2. IF

3. Hemagglutination inhibition test: some viruses can agglutinate red

cells of certain animals. This can be inhibited by addition of patient
serum containing specific Ab for the virus.

4. Neutralization test: (can be done on TC or in mice)

Neutralization Test in Mice

~ 58 ~
 Chapter 8

Laboratory Diagnosis of fungal infections

1- Sample: Skin scales, broken hair, nail pieces, sputum or any body fluid.
2- Direct microscopic examination of specimens: with KOH (10-20%)
preparation.

3- Culture of organism: On Sabaroud Dextrose Agar (SDA) aerobically at 37º c or

room temperature. Identification of growth by:
• Colony morphology
• Staining of colony using lactophenol cotton blue stain to see
morphological structure of fungi (as above)
4-DNA detection method: Using PCR or probe to detect fungal DNA

5-Serological tests: Either detection of fungal antigen or antibodies to fungal

antigen in patient’s serum using ELISA or IF.

~ 59 ~
 I. Candida

Gram Stained Smear of Candida Gram Film of Candida from

Showing Budding Cells and Culture
Pseudohyphae [Link]
[Link]

Candida on Sabouraud’s Agar Oral Candidiasis

[Link] [Link]

~ 60 ~
 II. Cryptococcus

Cryptococcus Capsule Stained by India Ink

[Link]

Cryptococcus on Sabouraud’s Agar

[Link]

~ 61 ~
 III. Dermatophytes

Hyphael Element by Direct Microscopic Examination

[Link]

Lactophenol Blue Mount of Infected Hairs Showing

"Small Spored" Ectothrix Invasion by M. Canis
[Link]

~ 62 ~
Macroconidia and Microconidia of Dermatophytes
[Link]

Tinea capitis Tinea corporis

[Link]

~ 63 ~
 IV. Aspergillus

Aspergillus Film Showing Radiating Chain Conidia

[Link]

Culture of Aspergillus fumigatus

[Link]

~ 64 ~