MICROBIOLOGY LAB MANUAL

Offered to
THREE-YEAR HONOURS DEGREE COURSE OF
STUDIES

UNIVERSITY OF CALCUTTA

CBCS SYLLABUS

2018

CU Roll No.:

CU Regn. No.:
CC-14: MEDICAL MICROBIOLOGY

(PRACTICAL)

SEMESTER –6

MCB-A-CC-6-14-P
 INDEX

Serial Name of the experiment Date Signature

No.

1 Identify bacteria (any three E. coli,

Salmonella sp., Pseudomonas sp.,
Staphylococcus sp., Bacillus sp.) using
laboratory strains on the basis of cultural,
morphological & biochemical
characteristics : IMViC, TSI, Nitrate
Reduction, Urease production, Catalase tests

2 Study of composition & use of important

differential media for identification of
bacteria :
EMB agar, McConkey agar, Mannitol Salt
agar, Deoxycholate-citrate agar, TCBS

3 Study of bacterial flora of skin by swab

method

4 Perform anti-bacterial sensitivity by Kirby-

Bauer method

5 Determination of Minimal Inhibitory

Concentration (MIC) of an antibiotic

6 Study symptoms of the diseases with the

help of photographs :
Polio, Anthrax, Herpes, Chicken pox, HPV
warts, AIDS (Candidiasis),
Dermatomycoses (Ring worms)
1. Identify bacteria (any three of E. coli, Salmonella, Pseudomonas,
Staphylococcus, Bacillus) using laboratory strains on the basis of
cultural, morphological and biochemical characteristics: IMViC,
TSI, nitrate reduction, urease production and catalase tests.

INTRODUCTION:
Bacteria are identified routinely by morphological and cultural characteristics and by different
biochemical tests. In case of clinically relevant microbes, proper identification is of utmost
importance because of various reasons like diagnosis, differences in pathogenicity, the
necessity to characterize a disease outbreak, for treatment and control, for therapeutic
applications etc.

Morphological Characteristics
Both wet-mounted and properly stained bacterial cell suspensions can yield a great deal of
information. These simple tests can indicate the Gram reaction of the organism; whether it is
acid-fast; its motility; the arrangement of its flagella; the presence of spores, capsules, and
inclusion bodies; and, of course, its shape. This information often can allow identification of
an organism to the genus level, or can minimize the possibility that it belongs to one or another
group. Bacteria can be classified by direct examination with the light microscope according to
their morphology and arrangement. The basic morphologies are: coccus (sphere), bacillus
(rod), spiral, comma shaped etc. Arrangements include pairs, tetrads, clusters, chains and
palisades.

Biochemical Characteristics
Just as human beings possess a characteristic and specific set of fingerprints, microorganisms
all have their own identifying biochemical characteristics. Most bacteria are identified and
classified largely on the basis of their reactions in a series of biochemical tests. Some tests are
used routinely for many groups of bacteria (oxidase, nitrate reduction, amino acid degrading
enzymes, fermentation of carbohydrates etc.); others are restricted to a single family, genus, or
species (e.g., coagulase test for staphylococci).

Cultural characteristics:
Cultural characteristic or colony morphology of bacteria is one of the important aspects applied
in identification of bacteria. Cultural characteristics describe various features of growth of
bacteria depending on the medium used (solid media as in agar plates, liquid media, whether
in slant/stab culture). They include colony shape, size, surface, elevation, margin, opacity,
pigmentation, texture, amount of growth, pattern of growth when in slant (diffused/filiform/
rhizoid/beaded), etc. Colony characteristics are dependent on growth conditions (temperature,
pH, incubation period) and on the type of media used.

Growth on solid media

Size – in mm
Shape – Round, irregular, rhizoid, spindle
Elevation – Flat, raised, convex, umbonate
Surface – Smooth, rough, papillate, ringed, etc
Margins – Entire, wavy, lobate, erose, curled, filamentous
 Opacity – Opaque, translucent, transparent
Pigmentation if any

Growth on slant
Amount – Scanty, moderate, abundant
Form – Spreading, filiform, rhizoid

Growth in stab
Amount – Scanty, moderate, abundant
Form – Papillate, filiform, beaded, arborescent
Surface growth– Absent, present, colouration
Liquification – forming layer, like cup, like turnip, like funnel, tubular

Growth in liquid media

Amount – Scanty, moderate, abundant
Surface growth– Absent, present, ring, pellicle (thin, thick, smooth, granular)
Deposit -Absent, present (slight, moderate, abundant, powdery, viscid)
Turbidity – Absent, present
Odour – Absent, present

Observations

1. Gram characteristics & morphology

Organism Gram Nature Morphology

E. coli ‘-ve’ Long rods
Klebsiella sp. ‘-ve’ Short rods
Staphylococcus sp. ‘+ve’ Spherical
Pseudomonas sp. ‘-ve’ Rod shaped

2. Growth Characteristics on Solid media

Organism Size Shape Elevation Surface Margin Opacity Colour Count

(mm)
E. coli 1 Round Flat Smooth Entire Translucent White 216 +
fused
Klebsiella sp. 1-5 Round Convex Smooth Entire Translucent White 255+
fused
Staphylococcus 1-2 Round Raised Smooth Entire Opaque Yellow 127 +
sp. fused
Pseudomonas 1-3 Round Flat Smooth Entire Opaque Off- 118 +
sp. white fused
 3. Growth Characteristics on Agar Slant

Organism Amount Form

E. coli Abundant Spreading
Klebsiella sp. Moderate Spreading
Staphylococcus sp. Abundant Spreading
Pseudomonas sp. Abundant Spreading

4. Growth characteristics on Stab Culture

Organism Amount Form Surface Growth Liquefaction Colour

E. coli Abundant Papillate Present Absent White
Klebsiella sp. Abundant Papillate Present Absent White
Staphylococcus sp. Abundant Filiform Present Absent Yellow
Pseudomonas sp. Scanty Filiform Present Absent White

5. Growth characteristics in Liquid Media

Organism Amount Surface Deposit Turbidity Odour

Growth
E. coli Abundant Absent Absent ‘+ve’ Intense
rotten smell
Klebsiella sp. Moderate Ring +; ‘+ve’ Rotten egg
Moderate
Staphylococcus sp. Scanty Ring +; ‘+ve’ ‘-ve’
Abundant
Pseudomonas sp. Abundant Granular +; ‘+ve’ Rotten egg
pellicle Moderate
 IMViC

Identification of the type of enteric/ coliform bacteria

Escherichia coli and Enterobacter aerogenes are the faecal and non-faecal coliforms
respectively. They closely resemble each other in morphological and cultural characteristics.
The following biochemical tests are performed to differentiate them. Tests are collectively
known as IMViC from the first letters of individual test.
I- Indole test
M- Methyl Red test
V- Voges Proskauer Test
C- Citrate utilization test

Coliforms are grouped into two types- Faecal and non-faecal based on the following
characteristics:

Indole Test
This test is performed to differentiate between faecal and non-faecal coliforms.

Principle:

Tryptophan is an essential amino acid that can undergo oxidation by way of enzymatic
activities of some bacteria. Conversion of tryptophan into metabolic products is mediated by
the enzyme tryptophanase. This ability to hydrolyse tryptophan with the production of indole
is not a characteristic of all microorganisms and therefore serves as a biochemical marker.
Tryptone broth is used for this test. Tryptone is a good substrate for indole production because
of its high tryptophan content. The presence of indole is detectable by Kovac’s reagent which
produces a cherry red complex layer. This reagent is composed of para-
dimethylaminobenzaldehyde, butanol and HCl. Indole becomes separated from the medium
into the reagent layer by the acidified butanol component and forms a complex compound of
cherry red colour with para-dimethylaminobenzaldehyde which indicates a positive test and its
absence denotes a negative test.
Procedure:

Composition of 1 lit Tryptone broth: (Ingredients gms / litre) ~

[https://www.himedialabs.com/TD/M463.pdf]
Tryptone: 10.000
Sodium chloride: 5.000
Final pH (at 25°C): 7.5±0.2

Steps:
1. Make the test tubes and broth sterile.
2. Inoculate the broth with the experimental colony from EMB agar plate.
3. Incubate the inoculated broth for 24h at 37oC.
4. After 24h, add Kovac’s reagent to the broth culture and observe the changes.
Record your observations:

Results:
Methyl red Test
This test is performed to differentiate between a faecal and non-faecal coliform.

Principle:

Hexose glucose is the major substrate oxidized by all enteric bacteria for energy production. In
this test pH indicator methyl red detects the presence of large concentration of acid and gas
products. Both faecal and non-faecal coliforms produce organic acid and other related products
during their early incubation period. In late phase E. aerogenes enzymatically converts these
acidic products to non-acidic components like 2,3- butanediol or acetoin (
acetylmethylcarbinol) resulting in the elevation of culture pH to approximately 6. Methyl red
indicator in the pH range of 4 will turn red which indicates the positive test and at pH 6 the
indicator turns yellow and is the negative test.

Procedure:

1. Prepare MRVP broth (5ml per tube) and autoclave it.

2. Inoculate the broth with the experimental colony from EMB agar plate.
3. Incubate the inoculated broth for 24h at 37oC.
4. After 24h add methyl red indicator drop wise to the broth culture and observe the changes.
Record your observations:

Results:
Voges Proskauer test
This test is performed to differentiate between a faecal and non-faecal coliform.

Principle:
Voges Proskauer (VP) test determines the capacity of some organisms to produce non-
acidic/neutral products such as acetylmethylcarbinol, from organic acids resulting from
glucose metabolism. The reagents used in this test are Barrit’s reagent A (Alcoholic alpha-
naphthol) and Barrit’s reagent B (40% KOH solution). Detection of acetylmethylcarbinol
requires that the end product will be oxidized to a diacetyl compound. This reaction will occur
in presence of alpha naphthol catalyst and a guanidine group (comes from peptone component
of MR-VP medium used). As a result a pink complex is formed imparting a rose colour to the
medium. This represents a positive test and its absence indicates a negative test.

Procedure:
1. Prepare MRVP broth (5ml per tube) and autoclave it.
2. Inoculate the broth with the experimental colony from EMB agar plate.
3. Incubate the inoculated broth for 24h at 37oC.
4. After 24h add Barrit’s reagent drop wise to the broth culture and observe the changes.
Record your observations:

Results:
Citrate utilization test
This test is performed to differentiate between a faecal and non-faecal coliform.

Principle:

Simmon's Citrate medium contains citrate as the only carbon source and ammonium ion as the
only nitrogen source. Bacteria that can utilize citrate have proteins (citrate permease) that bring
it across the cell membrane to metabolize it. Most bacteria cells can also bring ammonium ions
inside the cell and utilize them for protein and nucleic acid synthesis.
This test is often discussed as being positive if the medium turns blue anywhere in the tube and
negative if it remains green. This colour change is caused by a pH shift. Here, there are actually
two tests going on at once: whether citrate can be used as a carbon source and whether
ammonium can be used as a nitrogen source. Trisodium citrate present in the medium forms
sodium (3Na+) and citrate (C6H5O7-3) ions. If the cell can, it will bring citrate ions into the cell
to metabolize them to acetate and pyruvate. Most bacteria can metabolize pyruvate, and some
can metabolize acetate, to different products, depending on the pathway used, but usually to
carbon dioxide and water. The metabolism of citrate removes a weak base from the media. The
carbon dioxide that is generated combines with sodium and water to form sodium carbonate,
an alkaline product. The presence of sodium carbonate changes the bromothymol blue indicator
incorporated into the medium from green to blue. But if acetate is not metabolized, it will build
up and replace the weak base, so the net change in pH is uncertain.
Ammonium phosphate in the medium forms ammonium (NH4+) and dihydrogen phosphate
(H2PO4-) ions (note: H2PO4- will also partially disassociate into H+ + HPO4-2). If the cell can
reproduce by utilizing citrate, it will likely also utilize ammonium salts, removing it from the
medium to make nitrogenous biomolecules. Leaving the phosphate ions behind, which are
weak bases, and removing the ammonium ions, which are weak acids, will increase the
alkalinity of the medium and change the colour to blue.
Procedure:
1. Prepare sterile Simmon's Citrate agar slants.
2. Inoculate the slants with test organism from EMB agar plates.
3. Incubate the slants for 24-48h at 35oC

Composition per lit:

Record your observations:

Organism E. coli Klebsiella sp. Staphylococcus Pseudomonas

sp. sp.
Citrate
utilization Test ‘-ve’ ‘+ve’ ‘-ve’ ‘+ve’
Result

Result:
TRIPLE SUGAR–IRON (TSI) AGAR TEST

Principle:
Different types of bacteria can ferment different types (but not all types) of sugars. Thus, the
sugars, which a bacteria can ferment and the sugars, which it cannot ferment, is the
characteristic of the particular bacteria and thus an important criterion for its identification.
The Triple Sugar Iron (TSI) test is designed to differentiate among the different groups or
genera of the Enterobacteriaceae, which are all gram-negative bacilli capable of fermenting
glucose with the production of acid, and to distinguish the Enterobacteriaceae from other gram-
negative intestinal bacilli. This differentiation is made on the basis of differences in
carbohydrate fermentation patterns and hydrogen sulphide production by the various groups of
intestinal organisms.
To facilitate observation of carbohydrate utilization patterns, the TSI agar slants contain lactose
and sucrose in 1% concentrations and glucose (dextrose) in a concentration of 0.1%, which
permits detection of the utilization of this substrate only.
An agar slant of a special medium with multiple sugars, a pH-sensitive dye (phenol red), as
well as sodium thiosulfate and ferrous sulphate (or ferrous ammonium sulphate) is used for
carrying out the test. All of these ingredients are mixed together and allowed solidification at
an angle, thus resulting in a slanted shape of this medium that provides an array of surfaces
with either an aerobic or an anaerobic environment, under which fermentation patterns of
organisms are determined. The slant is inoculated by means of a stab-and-streak procedure.
Bacteria that ferment any of the three sugars in the medium will produce by-products, which
are usually acids, and will change the colour of the red pH-sensitive dye (phenol red) to a
yellow colour. Position of the colour change distinguishes the acid production associated with
glucose fermentation from the acidic by-products of lactose or sucrose fermentation. Many
bacteria that can ferment sugars in the anaerobic butt of the tube are enterobacteria. Some
bacteria (like Salmonella) utilize thiosulfate anion as a terminal electron acceptor, reducing it
to sulphide. If this occurs, the newly formed hydrogen sulphide reacts with ferrous sulphate in
the medium to form ferrous sulphide, which is visible as a black precipitate. Under anaerobic
conditions (as towards the bottom of the tube) some bacteria use thiosulfate as an electron
acceptor and reduce it to hydrogen gas. This is not very soluble and may accumulate as bubbles
along the inoculation track, between the agar and the glass, or in the fluid which accumulates
at the bottom of the slant. Hydrogen production may lift the agar from the butt of the tube or
fracture the agar (crack the agar). On the other hand, carbon dioxide, if produced, may not show
as bubbles because it is far more soluble in the medium.
Composition of Triple Sugar Iron Agar (TSI Agar) :

Ingredients Grams/litre

Beef extract 3.0 g

Yeast extract 3.0 g

Peptone 20.0 g

Glucose 1.0 g

Lactose 10.0 g

Sucrose 10.0 g

Ferrous sulphate or ferrous ammonium sulphate 0.2 g

NaCl 5.0 g

Sodium thiosulfate 0.3 g

Phenol red 0.024 g

Agar 13.0 g

Distilled water 1,000 ml (final volume)

Combine the ingredients, and adjust the pH to 7.3. Boil to dissolve the agar and dispense into
tubes. Sterilize by autoclaving. Cool in a slanted position to give a 2.5 cm butt and a 3.8 cm
slant.

TSI agar is also available commercially.

NOTE:

 TSI tube contains a butt region (poorly oxygenated area on the bottom) and a slant
surface (angled well-oxygenated area on the top).

 Phenol red: Indicator of acidification (It is yellow in acidic condition and red under
alkaline conditions).

 Media also contains peptone which acts as a source of nitrogen. Whenever peptone is
utilized under aerobic condition, ammonia is produced.
Procedure for Triple Sugar Iron Agar (TSI) Test:

With a sterilized straight inoculation needle touch the top of a well-isolated colony. Using
aseptic technique, inoculate each experimental organism into its appropriately labelled tube by
means of a stab-and-streak method. Inoculate TSI agar by first stabbing through the centre of
the medium to the bottom of the tube and then streaking on the surface of the agar slant. Leave
the cap on loosely and incubate the tube at 35-37°C for 18 to 24 hours. One tube should be kept
as control.

Following incubation, determine the fermentative activities of the organisms. Examine the
colour of both the butt and slant of all agar slant cultures. Based on your observations,
determine the type of reaction that has taken place (acid, alkaline, or none) and the carbohydrate
that has been fermented (dextrose, lactose, and/or sucrose, all, or none) in each culture. Also,
examine all cultures for the presence or absence of blackening within the medium. Based on
your observations, determine whether or not each organism was capable of H2S production.

Record your observations and results.

Interpretation of Triple Sugar Iron Agar Test:

If lactose or sucrose (both present in higher concentrations) gets fermented, a large amount
of acid is produced, which turns the phenol red indicator yellow both in the butt and in the
slant due to continued fermentative activities with maintenance of an acid reaction (in both
slant and butt). Some organisms generate gases, which produces bubbles/cracks on the
medium.

If lactose is not fermented but the small amount of glucose is, the oxygen-deficient butt will be
yellow (butt has comparatively more glucose than slant), but on the slant surface the small
amount of acid produced will be oxidized rapidly to carbon dioxide and water and the slant
will be red (alkaline or neutral pH). The peptones in the medium are also used in the production
of alkali. In the butt the acid reaction is maintained because of reduced oxygen tension and
slower growth of the organisms. Thus, an alkaline slant (red) and an acid butt (yellow) with
or without gas production (breaks in the agar butt) indicate that only glucose
fermentation has occurred.

If neither lactose/sucrose nor glucose is fermented, both the butt and the slant will be red.
The slant can become a deeper red-purple (more alkaline) as a result of the production of
ammonia from the oxidative deamination of amino acids (as peptone is a major constituent of
TSI agar). If only aerobic degradation of peptones occurs, the alkaline reaction is evidenced
only on the slant surface. If there is both aerobic and anaerobic utilization of peptone, the
alkaline reaction is present on the slant and the butt. But no carbohydrate fermentation has
occurred.

If H2S is produced, the black colour of ferrous sulphide is seen. The TSI agar medium also
contains sodium thiosulfate, a substrate for hydrogen sulphide (H2S) production, and ferrous
sulphate for detection of this colourless end product. Following incubation, only cultures of
organisms capable of producing H2S will show an extensive blackening in the butt because of
the precipitation of the insoluble ferrous sulphide.

Uses of the test:

The test is used primarily to differentiate members of the Enterobacteriaceae family from other
gram-negative rods. It is also used in the differentiation among Enterobacteriaceae on the basis
of their sugar fermentation patterns.

Image source: Clark College

Some example of Triple Sugar Iron (TSI) Agar Reactions:

Name of the organism Slant Butt Gas H2S

Escherichia, Klebsiella,
Enterobacter Acid (A) Acid (A) Pos (+) Neg (-)

Shigella, Serratia Alkaline (K) Acid (A) Neg (-) Neg (-)

Salmonella, Proteus Alkaline (K) Acid (A) Pos (+) Pos (+)

Pseudomonas
Alkaline (K) Alkaline (K) Neg (-) Neg (-)
A few precautionary measures:

 It is important to stab the butt of the medium. Failure to stab the butt invalidates this
test. The integrity of the agar must be maintained when stabbing. Caps must be loosened
during this test or erroneous results will occur.

 It is absolutely essential to observe the cultures within 18 to 24 hours following

incubation. Doing so will ensure that the carbohydrate substrates have not been depleted and
that degradation of peptones yielding alkaline end products has not taken place.

 An organism that produces hydrogen sulphide may mask acid production in the butt of
the medium. However, hydrogen sulphide production requires an acid environment, thus the
butt portion should be considered acid.

Record your observations:

Name of the organism Slant Butt Gas H2S

Escherichia coli Acid (A) Acid (A) Pos (+) Neg (-)

Klebsiella sp. Acid (A) Acid (A) Pos (+) Neg (-)

Salmonella sp. Alkaline (K) Acid (A) Pos (+) Pos (+)
Pseudomonas sp. Alkaline (K) Alkaline (K) Neg (-) Neg (-)
 NITRATE REDUCTION TEST
Principle:

Nitrate reduction test is used for the differentiation of members of Enterobacteriaceae on the
basis of their ability to produce nitrate reductase enzyme that hydrolyses nitrate to nitrite which
may then again be converted to various nitrogen products like nitrogen oxide, nitrous oxide
and ammonia depending on the enzyme system of the organism and the atmosphere in which
it is growing. Such reduction of nitrates by some aerobic and facultative anaerobic
microorganisms occurs in the absence of molecular oxygen, and is an anaerobic process. In
these organisms, anaerobic respiration is an oxidative process whereby the cell uses inorganic
substances such as nitrates (NO3-) or sulfates (SO42-) to supply oxygen that is subsequently
utilized as a final hydrogen acceptor during energy formation. The biochemical transformation
may be visualized as follows:

Some organisms possess the enzymatic capacity to act further on nitrites to reduce them to
ammonia (NH3+) or molecular nitrogen (N2). These reactions may be described as follows:

Nitrate reduction can be determined by cultivating organisms in a nitrate broth medium. The
medium is basically a nutrient broth supplemented with 0.1% potassium nitrate (KNO3) as the
nitrate substrate. In addition, the medium is made into a semi-solid by the addition of 0.1%
agar. The semi-solidity impedes the diffusion of oxygen into the medium, thereby favouring
the anaerobic requirement necessary for nitrate reduction. Following incubation of the cultures,
an organism’s ability to reduce nitrates to nitrites is determined by the addition of two reagents:
Solution A, which is sulfanilic acid, followed by Solution B, which is α-naphthylamine.
 Note: This should not be confused with Barritt’s reagent.

Following reduction, the addition of Solutions A and B will produce an immediate

cherry red colour, if nitrite is present. This is considered a positive result.

Cultures not producing a colour change suggest one of two possibilities:

(1) nitrates were not reduced by the organism, or

(2) the organism possessed such potent nitrate reductase enzymes that nitrates were
rapidly reduced beyond nitrites to ammonia or even molecular nitrogen.

To determine whether or not nitrates were reduced past the nitrite stage, a small amount of zinc
powder is added to the basically colourless cultures already containing Solutions A and B. Zinc
reduces nitrates to nitrites. The development of red colour therefore verifies that nitrates were
not reduced to nitrites by the organism. If nitrates were not reduced, a negative nitrate reduction
reaction has occurred. If the addition of zinc does not produce a colour change, the nitrates in
the medium were reduced beyond nitrites to ammonia or nitrogen gas. This is a positive
reaction.

Requirements:

Trypticase-nitrate broth can be used to detect an organism’s ability to reduce nitrate to nitrite
or a further reduced nitrogenous compound such as nitrous oxide or nitrogen gas.

 Trypticase nitrate broth (pH 7.2): [recipe from Cappuccino J and Sherman N.
Microbiology: A Laboratory Manual. 10th edition.]

For 1 litre: Trypticase 20.0gm, Disodium phosphate 2.0gm, Dextrose 1.0gm, Agar 1.0gm,
Potassium nitrate 1.0gm; distilled water to make up final volume.

 Solution A (sulfanilic acid), Solution B (α-naphthylamine), and zinc powder.

Procedure:

1. Using aseptic technique, inoculate each experimental organism into its appropriately
labelled tube by means of a loop inoculation. One tube should serve as a control.

2. Incubate all cultures for 24 to 48 hours at 37°C.

3. Add five drops of Solution A and then five drops of Solution B to all nitrate broth
cultures.

4. Observe and record whether or not a red coloration develops in each of the cultures.

5. Add a minute quantity of zinc to the cultures in which no red colour developed. Observe
and record whether or not red coloration develops now.

Uses:

This test is used to identify intestinal bacteria that are able to reduce nitrates to nitrites. Also,
it can differentiate Mycobacterium tuberculosis from non-tubercle Mycobacterium, since M.
tuberculosis is the only Mycobacterium species with this capacity.

Record your observation :

Bacteria Red Coloration Red Coloration Nitrate End product
with Solutions A with Zinc Reduction
and B
(+) or (−) (+) or (−)
(+) or (−)
 CATALASE TEST
Principle:

During aerobic respiration, microorganisms produce hydrogen peroxide and, in some cases,
an extremely toxic superoxide. Accumulation of these substances will result in death of the
organism unless they can be enzymatically degraded. These substances are produced when
aerobes, facultative anaerobes, and microaerophiles use the aerobic respiratory pathway, in
which oxygen is the final electron acceptor, during degradation of carbohydrates for energy
production. Organisms capable of producing catalase rapidly degrade hydrogen peroxide as
illustrated:

Aerobic organisms that lack catalase can degrade especially toxic superoxide using the enzyme
superoxide dismutase; the end product of a superoxide dismutase is H2O2, but this is less toxic
to the bacterial cells than are the superoxide

Catalase production can be determined by adding 3% H2O2 to an appropriately incubated test

culture. If catalase is present, the chemical reaction mentioned is indicated by bubbles of free
oxygen gas. This is a positive catalase test; the absence of bubble formation is a negative
catalase test.

The catalase test can be done using (a) the tube method, (b) the plate method, and (c) slide
method.

Two of these are described below:

 Tube Method

1. Using aseptic technique, inoculate each experimental organism into its appropriately labelled
tube by means of a streak inoculation. One tube will serve as a control.

2. Incubate all cultures for 24 to 48 hours at 37°C.

3. Allow three or four drops of 3% hydrogen peroxide to flow over the entire surface of each
slant culture.

4. Examine each culture for the presence or absence of bubbling or foaming. Record your
results.
  Slide Method

1. Label slides with the names of the organisms.

2. Using a sterile loop, collect a small sample of the first organism from the culture tube and
transfer it to the appropriately labelled slide.

3. Place the slide in a Petri dish.

4. Place one drop of 3% hydrogen peroxide on the sample. Do not mix. Place the cover on the
Petri dish to contain any aerosols.

5. Observe for immediate presence of bubble formation (within 5-10 seconds). A positive result
is the rapid evolution of oxygen, as evidenced by profuse bubbling. A negative result is no
bubbles or only a few scattered bubbles. Record your results.

6. Repeat Steps 2 through 5 for the remaining test organisms.

Record your observations:

Bacteria Presence or absence of Catalase production

bubbling (+) or (−)

Note:

Catalase is an enzyme, which is produced by microorganisms that live in oxygenated

environments to neutralize toxic forms of oxygen metabolites. The catalase enzyme neutralizes
the bactericidal effects of hydrogen peroxide and protects them. Anaerobes generally lack the
catalase enzyme. The inability of strict anaerobes to synthesize catalase, peroxidase, or
superoxide dismutase may explain why oxygen is poisonous to these microorganisms. In the
absence of these enzymes, the toxic concentration of H2O2 cannot be degraded when these
organisms are cultivated in the presence of oxygen.

The hydrogen peroxide reagent must be tested with positive and negative control organisms
each day or immediately before unknown bacteria are tested.

A. Positive control: Staphylococcus aureus

B. Negative control: Streptococcus species

Uses:

This test is used to identify organisms that produce the enzyme catalase which can break down
hydrogen peroxide. The catalase test is used for the biochemical differentiation of catalase-
positive Staphylococci and catalase-negative Streptococci, as well as members of the
Enterobacteriaceae.
 UREASE TEST
Principle:

The urease test is primarily used to distinguish the small number of urease-positive enteric.
Many enteric can degrade urea but only a few are termed rapid urease positive organisms.
While part of the normal flora, these commensals have been identified as opportunistic
pathogens.

Urease is especially helpful in the identification of Proteus vulgaris. Although other organisms
may produce urease, their action on the substrate urea tends to be slower than that seen with
Proteus species. Therefore, this test serves to rapidly distinguish members of this genus from
other non–lactose-fermenting enteric microorganisms.

Urease is a hydrolytic enzyme that attacks the nitrogen and carbon bond in amide compounds
such as urea and forms the alkaline end product ammonia. The presence of urease is detectable
when the organisms are grown in a urea broth medium containing the pH indicator phenol red.
As the substrate urea is split into its products, the presence of ammonia creates an alkaline
environment that causes the phenol red to turn to a deep pink. This is a positive reaction for the
presence of urease. Failure of a deep pink colour to develop is evidence of a negative reaction.

Media:

1 litre of Urea test broth is prepared by adding 20 g of urea, 9.5 g of Na2HPO4, 9.1 g of KH2PO4,
0.1g of yeast extract and 0.01 g of phenol red. Make up volume with distilled water. The pH
should be 6.8±0.2 at 25°C.

It is also available commercially.

It has to be filter sterilized and not heated.

Procedure:

1. Using aseptic technique, inoculate each experimental organism into its appropriately labelled
tube by means of loop inoculation. One tube should serve as a control.

2. Incubate cultures for 24 to 48 hours at 37°C.

3. Examine all urea broth cultures for colour and record your observation.
Bacteria Colour of Medium Urea Hydrolysis

(+) or (−)
2. Study of composition and use of important differential media for
identification of bacteria:
EMB Agar, McConkey agar, Mannitol salt agar, Deoxycholate
citrate agar, TCBS

i) EMB (Eosin methylene blue) agar~

It is a versatile solid differential medium originally developed by Levine, for the differentiation
of E. coli and Aerobacter aerogenes, for the rapid identification of Candida albicans and for
the identification of coagulase-positive Staphylococci.

Uses:

EMB, also known as "Levine's formulation" is a selective stain for Gram-negative bacteria and
are toxic to Gram-positive bacteria. It is used as selective and differential medium for the
isolation and differentiation of coliforms and fecal coliforms. EMB provides a colour indicator
distinguishing between organisms that ferment lactose (e.g., E. coli) and those that do not (e.g.,
Salmonella, Shigella). Organisms that ferment lactose display "nucleated colonies"…. colonies
with dark centres. Rapid lactose fermentation produces acids, which lower the pH. This
encourages dye absorption by the colonies, which are now coloured purple-black. Lactose non-
fermenters may increase the pH by deamination of proteins. This ensures that the dye is not
absorbed. The colonies will be colourless or light pink/lavender.

On EMB if E. coli is grown, it will give a distinctive metallic green sheen. Rapid fermentation
of lactose & production of strong acids (thus a rapid reduction in the pH of the EMB agar) act
as critical factors in the formation of the green metallic sheen.

EMB agar is used in water quality tests to distinguish coliforms and fecal coliforms that signal
possible pathogenic microbial contamination in water samples (presence of E. coli in the
river/water sample indicates the possibility of fecal contamination of water and the presence of
other pathogenic enteric). The medium is important in medical laboratories to distinguish gram-
negative pathogenic microbes in a short period of time.

Composition of the medium: per litre

Dissolve the above chemicals in distilled water. Adjust pH of the solution to 7.4. Add 15 gm
of Agar. Dissolve the agar by steaming in an autoclave for 30 min. (note: final volume should
be 1 lit).

Autoclave to sterilize the media. Use for pouring plates or for slants.
ii) MacConkey agar ~

It is a differential plating medium recommended for the detection and isolation of coliforms
and intestinal pathogens from stool, urine, water and other material.

Uses:

MacConkey agar is a selective and differential culture medium for bacteria. It is designed to
selectively isolate Gram-negative and enteric bacilli and differentiate them based on lactose
fermentation. Lactose fermenters turn red or pink on McConkey agar, and non-fermenters do
not change colour. The media inhibits growth of Gram-positive organisms with crystal violet
and bile salts, allowing for the selection and isolation of Gram-negative bacteria. The media
detects lactose fermentation by enteric bacteria with the pH indicator neutral red (turns pink if
the microbes are fermenting lactose).

Few examples of non-lactose fermenting bacteria are Salmonella, Proteus species, Yersinia,
Pseudomonas aeruginosa and Shigella. Examples of lactose fermenters include Escherichia
coli, Enterobacter and Klebsiella. Escherichia coli produce greater quantities of acid from
lactose than other coliform species. When this occurs, the medium surrounding the growth also
becomes pink because of the action of the acid that precipitates the bile salts, followed by
absorption of the neutral red.

Composition of the medium: per litre

There are many variations of MacConkey agar depending on the need. If the spreading or
swarming of Proteus species is not required, sodium chloride is omitted, otherwise added@
5gm per lit. Crystal violet at a concentration of 0.0001% (0.001 g per litre) is included when
needing to check if Gram-positive bacteria are inhibited. MacConkey with sorbitol is used to
isolate E. coli O157, an enteric pathogen.

Two variations are given below:

1.
2.
iii) Mannitol salt agar ~
Mannitol salt agar or MSA is a commonly used selective and differential growth medium that
encourages the growth of a group of certain bacteria while inhibiting the growth of others. This
medium is important in medical laboratories as one method of distinguishing pathogenic
microbes in a short period of time. It contains a high concentration (about 7.5–10%) of salt
(NaCl) which is inhibitory to most bacteria, making MSA selective against most Gram-
negative and selective for some Gram-positive bacteria (Staphylococcus, Enterococcus and
Micrococcaceae) that tolerate high salt concentrations. It is also a differential medium for
mannitol-fermenting staphylococci, as it contains carbohydrate mannitol and the pH indicator
phenol red. If an organism can ferment mannitol, an acidic by-product is formed that causes
the phenol red in the agar to turn yellow. Staphylococcus aureus produces yellow colonies with
yellow zones, whereas non-fermenter staphylococci produce small pink or red colonies with
no colour change to the medium. It is used for the selective isolation of presumptive pathogenic
Staphylococcus species.

Composition: per litre

v) Deoxycholate citrate agar~
Uses:

DCA medium is particularly useful for the isolation of organisms that cause bacillary
dysentery, Salmonella strains that cause food poisoning and Salmonella paratyphi. It is not so
selective for Salmonella typhi. Salmonella spp appear to be yellow or colourless colonies, often
with a dark centre. As there are many bacteria that also look like Salmonella on DCA, it is
widely recommended that more selective agars are used for the identification of Salmonella,
namely xylose lysine deoxycholate (XLD) agar.

Sodium deoxycholate at pH 7.3 to 7.5 is inhibitory for gram-positive bacteria. Citrate salts, in
the concentration included in the formulation, are inhibitory to gram-positive bacteria and most
other normal intestinal organisms. It is thus a selective and differential medium, used
commonly for the isolation of enteric pathogens.

Composition:
One litre of deoxycholate citrate agar contains :

Sodium citrate 20 grams

Agar 13.5 gm

Lactose 10 grams

Solids from meat infusion (HI solids) 10 grams

Peptic digest of animal tissue (Proteose peptone) 10 grams

Sodium deoxycholate 5 grams

Ferric ammonium citrate 2 gram

Neutral red 20 mg

This growth medium is heat-sensitive and should be poured and cooled as soon as possible
after addition of the deoxycholate, otherwise it tends to become very soft and difficult to
 handle. It has a pH of approximately 7.3, and when poured and cooled, appears light to
dark pink in colour.

DO NOT AUTOCLAVE!!

NOTE:

HI solids form a source of carbon and nitrogen and this ingredient is used because the inhibition
of coliforms produced is greater than when an extract or simple peptone is used. Proteose
peptone provides carbon, nitrogen, vitamins and minerals. Coliform bacteria and gram-positive
bacteria are inhibited or greatly suppressed due to sodium deoxycholate, sodium citrate and
ferric ammonium citrate. Lactose helps in differentiating enteric bacilli, as lactose fermenters
produce red colonies while lactose non-fermenters produce colourless colonies. Coliform
bacteria, if present form pink colonies on this medium. The degradation of lactose causes
acidification of the medium surrounding the relevant colonies and the pH indicator neutral red
changes its colour to red. These colonies usually are also surrounded by a turbid zone of
precipitated deoxycholic acid due to acidification of the medium. Sodium deoxycholate
combines with neutral red in an acidic environment, causing the dye to go out of the solution
with the subsequent precipitation of deoxycholate. The reduction of ferric ammonium citrate
to iron sulfide is indicated by the formation of black iron sulfide. Salmonella and Shigella
species do not ferment lactose but Salmonella may produce H2S, forming colourless colonies
with or without black centres.
vi) TCBS~
Uses:

Thiosulfate-citrate-bile salts-sucrose agar, or TCBS agar, is a type of selective agar culture

plate that is used to isolate Vibrio species (V. cholerae and V. parahaemolyticus as well as
other Vibrio species).

TCBS agar contains high concentrations of sodium thiosulfate and sodium citrate to inhibit the
growth of Enterobacteriaceae. Inhibition of Gram-positive bacteria is achieved by the
incorporation of ox gall, which is a naturally occurring substance containing a mixture of bile
salts and sodium cholate, a pure bile salt. Sodium thiosulfate also serves as a sulfur source and
its presence, in combination with ferric citrate, allows for the easy detection of hydrogen
sulfide production. Saccharose (sucrose) is included as a fermentable carbohydrate for
metabolism by Vibrio species. The alkaline pH of the medium enhances the recovery of V.
cholerae and inhibits the growth of others. Thymol blue and bromothymol blue are included
as indicators of pH changes.

To be precise, TCBS agar is, in fact, both selective and differential. It is highly selective for
Vibrio species and differential due to the presence of sucrose and the dyes. Sucrose
fermentation produces acid, which converts the colour of bromothymol blue or thymol blue.
Two dyes rather than one make the medium produce an array of yellow, green, or blue thus
differentiating among various Vibrio species.

Composition (per litre):

 Yeast extract 5.0 g

 Proteose Peptone 10.0 g

 Sodium thiosulfate 10.0 g

 Sodium citrate 10.0 g

 Ox gall 5.0 g

 Sodium cholate 3.0 g

 Saccharose/ sucrose 20.0 g

 Sodium chloride 10.0 g

 Ferric citrate 1.0 g

 Bromothymol blue 0.04 g

 Thymol blue 0.04 g

 Agar 15.0 g

pH 8.6 ± 0.2 @ 25°C

Mix all ingredients and add distilled water to make volume 1 litre. Heat to boiling to dissolve
the medium completely. Do not autoclave. Cool to 45-50°C. Mix well and pour into sterile
Petri plates.

NOTE:

Yeast extract, bacteriological peptone: provides the nitrogen, vitamins, and amino acids in
TCBS agar

Sodium thiosulphate, Sodium citrate: selective agents, providing an alkaline pH to inhibit

Gram-positive organisms and suppress coliforms

Ox Bile: Bile salts inhibit growth of gram-positive microorganisms and coliforms

Sucrose: fermentable carbohydrate

Sodium chloride: provide optimum growth and metabolic activity of halophilic Vibrio spp.

Ferric citrate: Sodium thiosulfate is also a sulfur source, and acts with ferric citrate as an
indicator to detect hydrogen sulphide production.

Bromothymol blue/ thymol blue: pH indicator.

Agar: Solidifying agent.

Typical colony morphology:

V. cholerae: large (2 to 4 mm in diameter), slightly flattened, yellow colonies with opaque

centres and translucent peripheries. V. cholerae can ferment sucrose and this results in a
decrease in pH and production of yellow colonies

V. parahaemolyticus: Colonies with blue to green centres; does not ferment sucrose.

V. alginolyticus: Large yellow mucoidal colonies.

V. harveyi / V. fischeri: Greyish-green to bluish-green colonies which show luminescence in

dark. Older colonies fail to show bioluminescence.
3. Study of bacterial flora of skin by swab method
Introduction:

Skin provides good example of various microenvironments. The composition of the dermal
microflora varies from site to site according to the character of the microenvironment. A
different bacterial flora characterizes each of three regions of skin: (1) axilla, perineum, and
toe webs; (2) hand, face and trunk; and (3) upper arms and legs. Skin sites with partial occlusion
(axilla, perineum, and toe webs) harbour more microorganisms than do less occluded areas
(legs, arms, and trunk). These quantitative differences may relate to increased amount of
moisture, higher body temperature, and greater concentrations of skin surface lipids. The axilla,
perineum, and toe webs are more frequently colonized by Gram-negative bacilli than are drier
areas of the skin.

The number of bacteria on an individual's skin remains relatively constant; bacterial survival
and the extent of colonization probably depend partly on the exposure of skin to a particular
environment and partly on the innate and species-specific bactericidal activity in skin. Also, a
high degree of specificity is involved in the adherence of bacteria to epithelial surfaces.

The varied environment of the skin results in locally dense or sparse populations, with Gram-
positive organisms (e.g., staphylococci, micrococci, diphtheroids) usually predominating.

Principle:

Human skin is constantly covered with microorganisms both commensals and pathogens
depending on topography, environmental factors and host factors. Cultures from the skin have
frequently demonstrated bacteria such as Diphtheroids, Staphylococcus, Streptococcus
viridans, Streptococcus faecalis, Micrococcus, Corynebacteria, Brevibacteria,
Propionibacteria, gram positive aerobic spore-bearing bacilli, gram negative bacilli such as
Escherichia coli, Proteus and other intestinal organisms, non-pathogenic Mycobacteria and
fungi like Candida albicans. Hair frequently harbours Staphylococcus aureus and forms a
reservoir for cross-infection. Though these organisms are regarded as commensals in immune-
competent individuals, they can become pathogenic in immune-compromised persons
especially in hospitalised individuals. In recent years, Staphylococcus epidermidis is regarded
as an agent of hospital and community acquired infections. They act as causative agent of
bloodstream infections, urinary tract infections and surgical site infections. Another concern is
increasing incidence of drug resistance in these organisms. Penicillin resistant Staphylococcus
is seen in individuals working in hospitals. The skin of health care providers harbours
commensals as well as pathogens including drug resistant organisms due to exposure to
hospital environment, which may become sources of nosocomial infections. Thus study of skin
microbial flora becomes extremely important.
Procedure:

Swab samples have to be collected from various sites especially from the web space of hand
and foot. The bacterial colonies can be identified by Gram staining and biochemical reactions
and antibiotic susceptibility testing done according to standard protocols described in this
manual. Tests can be done for E. coli, Salmonella, Pseudomonas, Staphylococcus, Bacillus or
other pathogenic strains and observations recorded in tabular form.

Skin samples can be collected in the following ways.

 A dry sterile cotton-tip swab is rubbed on the suspicious skin site, for example, blistered
or dry skin lesions or pustules.
 A moist swab is taken from a mucosal surface, such as inside the mouth.

If the surface is dry, then moistening the tip of the swab with sterile saline will help bacteria
to adhere to the swab.
Sample the entire surface of the test area suspected to be infected using a zig-zag motion
whilst simultaneously rotating the swab between the fingers.

Place the swab in the culture medium. Maintain aseptic conditions.

Record your observations:

1(A) : Skin sample of

Construction worker
1(B) : Skin sample of
A B
Security

2(C) : Skin sample of

Street Vendor
2(D) : Skin sample of
C D
Chaat seller

3(E) : Skin sample of Face

3(F) : Skin sample of Face
E F

3
3
 4. Perform antibacterial sensitivity by Kirby-Bauer method
Principle:

Different chemotherapeutic agents vary in their scope of antimicrobial activity. Some have a
limited spectrum of activity, being effective against only one group of microorganisms. Others
exhibit broad-spectrum activity against a range of microorganisms. The drug susceptibilities of
many pathogenic bacteria are known, but it is sometimes necessary to test several agents to
determine the drug of choice. A standardized diffusion procedure with filter paper discs on
agar, known as the Kirby-Bauer method, is frequently used to determine the drug susceptibility
of bacteria isolated from infectious processes. This method allows the rapid determination of
the efficacy of a drug by measuring the diameter of the ‘zone of inhibition’ that result from
diffusion of the agent into the medium surrounding the disc.

In this procedure, filter-paper discs of uniform size are impregnated with specified
concentrations of different antibiotics and then placed on the surface of an agar plate that has
been seeded with the organism to be tested. The usual medium of choice is Mueller-Hinton
agar, with a pH of 7.2 to 7.4, which is poured into plates to a uniform depth of 5 mm and
refrigerated after solidification. Prior to use, the plates are transferred to an incubator at 37°C
for 10 to 20 minutes to dry off the moisture that develops on the agar surface. The plates are
then heavily inoculated with a standardized inoculum to ensure the confluent growth of the
organism. The discs are aseptically applied to the surface of the agar plate at well-spaced
intervals. Once applied, each disc is ensured to be in firm contact with the agar surface.

Following incubation, the plates are examined for the presence of growth inhibition, which is
indicated by a clear zone surrounding each disc. The susceptibility of an organism to a drug is
assessed by the size of this zone, which is affected by other variables such as:

1. The ability and rate of diffusion of the antibiotic into the medium and its interaction with the
test organism.

2. The number of organisms inoculated.

3. The growth rate of the organism.

A measurement of the diameter of the zone of inhibition in millimeters is made, and its size is
compared to that contained in a standardized chart, which is shown below. Based on this
comparison, the test organism is determined to be resistant, intermediate, or susceptible to the
antibiotic.
Requirements:

 Bacterial cultures

 Different antibiotics as supplied, e.g., Penicillin G, 10 μg; streptomycin, 10 μg;

tetracycline, 30 μg; chloramphenicol, 30 μg; gentamicin, 10 μg; vancomycin, 30 μg;
sulphanilamide, 300 μg.

 6-mm filter paper disks, forceps, Agar plates, alcohol, etc.

Composition of1 lit of MH agar is given below:

Procedure:

1. Place agar plates right side up in an incubator heated to 37°C for 10 to 20 minutes.

2. Label the covers of each of the agar plates with the name of the test organism to be
inoculated.

3. Using aseptic technique, inoculate all agar plates heavily with their respective test organisms.
4. Allow all culture plates to dry for about 5 minutes.

5. Apply the antibiotic discs onto the agar surface by putting the individual discs at equal
distances with forceps dipped in alcohol and flamed.

6. Gently press each disc down with sterile forceps to ensure that the discs adhere to the surface
of the agar. Note: Do not press the discs into the agar.

7. Incubate all plate cultures in an inverted position for 24 to 48 hours at 37°C.

8. Examine all plate cultures for the presence or absence of a zone of inhibition surrounding
each disc.

9. Using a ruler graduated in millimetres, carefully measure each zone of inhibition to the
nearest millimetre. Record your results.

10. Compare your results with the Table and determine the susceptibility of each test organism
to the chemotherapeutic agent. Record your results.

NOTE:
  zone of inhibition: This is an area of media where bacteria are unable to grow, due to
presence of a drug that impedes their growth.

 The size of the zone of inhibition of growth is influenced by the depth of the agar, since
the antimicrobial diffuses in three dimensions; thus, a shallow layer of agar will produce
a larger zone of inhibition than a deeper layer.

 MH agar is considered the best medium to use for routine susceptibility testing of non-
fastidious bacteria for the following reasons: It shows acceptable batch-to-batch
reproducibility for susceptibility testing. It is low in sulphonamide, trimethoprim, and
tetracycline inhibitors. It supports satisfactory growth of most non-fastidious
pathogens. A large body of data and experience has been collected concerning
susceptibility tests performed with this medium. Please note that the use of media other
than Mueller-Hinton agar may sometimes result in erroneous results.

[https://asm.org/getattachment/2594ce26-bd44-47f6-8287-0657aa9185ad/Kirby-Bauer-Disk-
Diffusion-Susceptibility-Test-Protocol-pdf.pdf]

Record your observations in tabular form (refer to table)

5. Determination of minimal inhibitory concentration (MIC) of an
antibiotic.

Principle:

Minimum Inhibitory Concentration (MIC): This is the lowest concentration of an antimicrobial

drug that prevents visible growth of a microorganism after overnight incubation with media.

The tube dilution test is the standard method for determining levels of resistance to
an antibiotic. Serial dilutions of the antibiotic are made in a liquid medium which is inoculated
with a standardized number of organisms and incubated for a prescribed time. The lowest
concentration (highest dilution) of antibiotic preventing appearance of turbidity is considered
to be the minimal inhibitory concentration (MIC). At this dilution the antibiotic
is bacteriostatic. Additionally, the minimal bactericidal concentration (MBC) can be
determined by sub-culturing the contents of the tubes onto antibiotic-free solid medium and
examining for bacterial growth.

Procedure:

 Number sterile capped test tubes 1 through 9. All of the following steps are carried out
using aseptic technique.

 Add 2.0 ml of antibiotic e.g., tetracycline solution (100 µg/ml) to the first tube.

 Add 1.0 ml of sterile broth to all other tubes.

 Transfer 1.0 ml from the first tube to the second tube.

 Using a separate pipette, mix the contents of this tube and transfer 1.0 ml to the third
tube.

 Continue dilutions in this manner to tube number 8, being certain to change pipettes
between tubes to prevent carryover of antibiotic on the external surface of the pipette.

 Remove 1.0 ml from tube 8 and discard it.

 The ninth tube, which serves as a control, receives no tetracycline.

 Suspend to an appropriate turbidity several colonies of the bacterial culture to be tested

in 5.0 ml of Mueller-Hinton broth to give a slightly turbid suspension.

 Dilute this suspension by aseptically pipetting 0.2 ml of the suspension into 40 ml of

Mueller-Hinton broth.
  Add 1.0 ml of the diluted culture suspension to each of the tubes. The final
concentration of tetracycline is now one-half of the original concentration in each
tube.

 Prepare another control tube containing only broth.

 Incubate all tubes at 35-37oC overnight.

 Examine tubes for visible signs of bacterial growth. The highest dilution without
growth is the minimal inhibitory concentration (MIC).

NOTE:

 The appropriate inoculum size for standard MIC is 10 4 to 105 CFU/ml.

 A lower MIC value indicates that less drug is required for inhibiting growth of the
organism; therefore, drugs with lower MIC scores are more effective antimicrobial
agents.

 By identifying appropriate drugs and their effective concentrations, MIC scores aid in
improving outcomes for patients and preventing evolution of drug-resistant microbial
strains.
Record your observations in tabular form.
 6. Study symptoms of the diseases with the help of photographs:
Polio, anthrax, herpes, chicken pox, HPV warts, AIDS
(candidiasis), dermatomycoses (ring worms)

Polio~

Poliomyelitis (Greek polios, gray, and myelos, marrow or spinal cord), polio, or infantile
paralysis is caused by poliovirus (species Human enterovirus C) and was first described in
England in 1789 as a "leg-wasting" disease of children, although a pictograph from ancient
Egypt clearly depicts a man with what appears to be polio.
The virus is an enterovirus - a transient inhabitant of the gastrointestinal tract-and a member of
the family Picornaviridae and as such is a non-enveloped, positive-strand RNA virus.
The virus enters the body through the mouth or nose, getting into the digestive and respiratory
(breathing) systems. It multiplies in the throat and intestines.
Generally there are either no symptoms or a brief illness characterized by fever, headache, sore
throat, fatigue, neck stiffness, pain in arms and legs, vomiting, and loss of appetite. The virus
sometimes enters the bloodstream, causing viremia. In most cases (more than 99%), the viremia
is transient and clinical disease does not result. However, in a minority of cases, the viremia
persists and the virus enters the central nervous system and causes paralytic polio. The virus
has a high affinity for anterior horn motor nerve cells of the spinal cord. Once inside these cells,
it multiplies and destroys the cells; this results in motor and muscle paralysis. About 5% to
10% of people paralyzed by polio die because they can’t use their muscles to breathe.
Anthrax~
Anthrax is an infection caused by the bacterium Bacillus anthracis. It can occur in four forms:
skin, lungs, intestinal, and injection.

Symptom onset may occur between one day to over two months after the infection is
contracted.

i) The skin form, Cutaneous anthrax, presents with a small blister with surrounding swelling
that often turns into a painless ulcer with a black center. It is the most common form (>90%
of anthrax cases). It is also the least dangerous form. Unlike bruises or most other lesions,
cutaneous anthrax infections normally do not cause pain. Nearby lymph nodes may become
infected, reddened, swollen, and painful. A scab forms over the lesion soon, and falls off in a
few weeks.

ii) The inhalation/ respiratory form initially presents with fever, chill, chest pain, fatigue,
cough, nausea and shortness of breath for the first few days. After that shortness of breath,
cough, and chest pain become more common; complaints not involving the chest such as
nausea, vomiting, altered mental status, sweats, and headache develop in one-third or more of
people. Upper respiratory tract symptoms occur in only a quarter of people, and muscle pains
are rare. It infects the lymph nodes in the chest first, rather than the lungs themselves, a
condition called “hemorrhagic mediastinitis”, causing bloody fluid to accumulate in the chest
cavity, therefore causing shortness of breath. The second (pneumonia) stage occurs when the
infection spreads from the lymph nodes to the lungs. Symptoms of the second stage develop
suddenly within hours or days after the first stage. Symptoms include high fever, extreme
shortness of breath, shock, and rapid death within 48 hours in fatal cases.
iii) The intestinal form presents with diarrhea which may contain blood, abdominal pains,
nausea, and vomiting. Occasional vomiting of blood can occur. Lesions have been found in the
intestines and in the mouth and throat. After the bacterium invades the gastrointestinal system,
it spreads to the bloodstream and throughout the body, while continuing to make toxins.

iv) The injection form presents with fever and an abscess at the site of drug injection. It may
have symptoms similar to cutaneous anthrax, but it may also have infection deep into the
muscle and spread faster.
Herpes~
Herpes simplex is a viral infection caused by the herpes simplex virus. Infections are
categorized based on the part of the body infected.

Once infected, a person will have herpes simplex virus for the rest of his or her life. When
inactive, the virus lies dormant in a group of nerve cells. While some people never develop any
symptoms from the virus, others will have periodic outbreaks of infections.

Oral herpes involves the face or mouth. It may result in small blisters in groups often called
cold sores or fever blisters which may be painful, or it may just cause a sore throat.

Genital herpes, often simply known as herpes, may have minimal symptoms or form blisters
that break open and result in small ulcers. These typically heal over two to four weeks. Tingling
or shooting pains may occur before the blisters appear.

Herpes cycles between periods of active disease followed by periods without symptoms. The
first episode is often more severe and may be associated with fever, muscle pains,
swollen lymph nodes and headaches. Over time, episodes of active disease decrease in
frequency and severity.

Other disorders caused by herpes simplex include: herpetic whitlow when it involves the
fingers, herpes of the eye, herpes infection of the brain and neonatal herpes when it affects a
newborn, among others.
Chicken pox~
Chickenpox is a very contagious infection caused by the varicella-zoster virus. It mainly affects
kids, but adults can get it, too. The tell-tale sign of chickenpox is a super-itchy skin rash with red
blisters. The rash first appears on the chest, back, and face, and then spreads over the entire body,
causing between 250 and 500 itchy blisters. Other typical symptoms that may begin to appear 1-2
days before rash include: fever, headache, tiredness, loss of appetite. Over the course of several
days, the blisters pop and start to leak. Then they crust and scab over before finally healing. Adults
have a higher risk for developing complications from chickenpox than children. It can also be
serious in babies, pregnant women, and people with a weakened immune system.
HPV warts~
HPV infection is a viral infection that commonly causes skin or mucous membrane growths
(warts). There are more than 100 varieties of human papillomavirus (HPV). Some types of
HPV infection cause warts, and some can cause different types of cancer, like cervical cancer.
In most cases, our body's immune system defeats an HPV infection before it creates warts.
When warts do appear, they vary in appearance depending on which kind of HPV is involved:

Genital warts: These appear as flat lesions, small cauliflower-like bumps or tiny stemlike
protrusions. In women, genital warts appear mostly on the vulva but can also occur near the
anus, on the cervix or in the vagina. In men, genital warts appear on the penis and scrotum or
around the anus. Genital warts rarely cause discomfort or pain, though they may itch or feel
tender.

Common warts: Common warts appear as rough, raised bumps and usually occur on the hands
and fingers. In most cases, common warts are simply unsightly, but they can also be painful or
susceptible to injury or bleeding.

Plantar warts: Plantar warts are hard, grainy growths that usually appear on the heels or balls
of your feet. These warts might cause discomfort.

Flat warts: Flat warts are flat-topped, slightly raised lesions. They can appear anywhere, but
children usually get them on the face and men tend to get them in the beard area. Women tend
to get them on the legs.
AIDS (candidiasis) ~
Candidiasis, commonly called thrush, is a fungal infection caused by strains of Candida, a type
of yeast. People living with HIV infection are more prone to candidiasis. It is not an uncommon
condition and generally manifests when a person's immune response is low.

The Candida yeast itself is present in most human beings, within the natural flora of the mouth
and digestive tract, as well as on the skin. It is only when changes to these systems occur
that Candida can actively thrive, usually manifesting with superficial infection. However,
when the immune system is severely compromised, as can happen with untreated
HIV, Candida can become invasive and spread throughout the body, causing severe illness and
even death.

Candidiasis is characterized by thick, white patches on the tongue, as well as other parts of the
mouth and throat. A sore throat and difficulty in swallowing can also accompany.

When candidiasis presents in the vagina, it is typically referred to as a yeast infection and is
characterized by a thick, cottage cheese-like discharge from the vagina. Vaginal burning,
itching, and soreness are commonly noted during outbreaks.

While less commonly seen, Candida infections can also occur on the skin; under the fingernails
or toenails; on the rectum, anus, or penis; or within the esophagus or pharynx.

Candida plaque can be scraped off from the tongue, walls of the mouth, or walls of the vagina,
revealing a sore, red, denuded patch underneath. The plaque is entirely odorless.

When an HIV infection is left untreated and a person's CD4 count dips beneath 200 cells/mL
(one of the official classifications of AIDS), the risk of invasive candidiasis is profoundly
increased. As a result, candidiasis of the esophagus, bronchi, trachea or lungs (but not the
mouth) is today classified as an AIDS-defining condition.
Dermatomycoses (ring worms)~
Dermatomycosis are mycotic diseases of skin caused by a few mycetes: dermatophytes, and
some opportunistic fungi as Malassezia, Candida (not C. albicans), Aspergillus, etc.
Dermatophytes are a group of closely related filamentous fungi that invade keratinized tissue
(skin, hair, nails) of humans and other animals and produce infection. Ringworm is a common
name for it. It’s called “ringworm” because it can cause a circular rash (shaped like a ring) that
is usually red and itchy. The fungi that cause this infection can live on skin, surfaces, and on
household items such as clothing, towels, and bedding.

Ringworm can affect skin on almost any part of the body as well as fingernails and toenails.
The symptoms of ringworm often depend on which part of the body is infected, but they
generally include:

 Itchy skin
 Ring-shaped rash
 Red, scaly, cracked skin
 Hair loss

Symptoms of ringworm by location on the body:

Feet (tinea pedis or “athlete’s foot”): The symptoms of ringworm on the feet include red,
swollen, peeling, itchy skin between the toes (especially between the pinky toe and the one
next to it). The sole and heel of the foot may also be affected. In severe cases, the skin on the
feet can blister.

Scalp (tinea capitis): Ringworm on the scalp usually looks like a scaly, itchy, red, circular
bald spot. The bald spot can grow in size and multiple spots might develop if the infection
spreads. Ringworm on the scalp is more common in children than it is in adults.

Groin (tinea cruris or “jock itch”): Ringworm on the groin looks like scaly, itchy, red spots,
usually on the inner sides of the skin folds of the thigh.

Beard (tinea barbae): Symptoms of ringworm on the beard include scaly, itchy, red spots
on the cheeks, chin, and upper neck. The spots might become crusted over or filled with pus,
and the affected hair might fall out.

https://www.cdc.gov/fungal/diseases/ringworm/symptoms.html
 7. Study of various stages of malarial parasite in RBCs using
permanent mounts.

The natural history of malaria involves cyclical infection of humans and

female Anopheles mosquitoes. In humans, the parasites grow and multiply first in the liver cells
and then in the red cells of the blood. In the blood, successive broods of parasites grow inside
the red cells and destroy them, releasing daughter parasites (“merozoites”) that continue the
cycle by invading other red cells. The blood stage parasites are those that cause the symptoms
of malaria.

Recognition of a malaria parasite: Malaria parasites take up Giemsa stain in a special way in
both thick and thin blood films.
Malaria parasites pass through a number of developmental stages. In all stages, however, the
same parts of the parasite will stain the same colour:

• Chromatin (part of the parasite nucleus) is usually round in shape and stains a deep red.
• Cytoplasm occurs in a number of forms, from a ring shape to a totally irregular shape. It
always stains blue, although the shade of blue may vary between the malaria species.

Stages of the malaria parasite in RBC:

The trophozoite stage: This stage is most commonly seen; it is often called the ring stage,
although it sometimes takes the form of an incomplete ring. Because the trophozoite stage is a
growing stage, the parasite within the red blood cell may vary in size from small to quite large.
Pigment appears as the parasite grows. Malaria pigment is a by-product of the growth or
metabolism of the parasite. It does not stain, but has a colour of its own, which may range from
pale yellow to dark brown or black.

The ring stages of malaria parasites

The schizont stage: At the schizont stage the malaria parasite starts to reproduce. This
reproduction is referred to as asexual because the parasite is neither male nor female but
reproduces itself by simple division. There are several obvious phases in this stage, ranging
from parasites with two chromatin pieces to parasites with a number of chromatin dots and
definite cytoplasm. The process of forming schizonts, which takes place in the liver and in
blood, is referred to as schizogony.

Stages of schizont growth

The gametocyte stage: The gametocyte stage is sexual in that the parasites become either male
or female in preparation for the next stage, which takes place in the stomach of the female
mosquito. Gametocytes may be either round or banana-shaped, depending on the species. The
way in which the parasite takes up the stain also helps to identify whether it is male
(microgametocyte) or female (macrogametocyte).

Male and female gametocytes

Finding P. falciparum P. vivax P. malariae P. ovale

RBC Not enlarged Enlarged Not enlarged Enlarged

Size

RBC Round, sometimes Round or oval, Round Round or oval, often

Shape crenated frequently bizarre fimbriated

RBC Normal, but may Normal to pale Normal Normal

Colour become darker; may
have a purple rim

Stipling Maurer’s spots, appear Schuffner’s dots, Ziemann’s Schuffner’s dots

as large red spots, appear as small red dots, few tiny (James’s dots).
loops and clefts; up to dots, numerous. dots, rarely Numerous small red
20 or fewer. detected dots.

Pigment Black or dark brown; Seen as a haze of Black or Intermediate

in asexual forms as fine golden brown brown coarse between P.
one or two masses; in granules scattered granules; vivax and P. malariae
gametocytes as about through the scattered
12 rods cytoplasm

Early Smallest, delicate; Relatively large; Compact; one Compact; one

trophozo sometimes two one chromatin dot, chromatin chromatin dot; single
ite (ring) chromatin dots; sometimes two; dot; single
multiple rings often two rings in
commonly found one cell

Schizont Medium size; Large; amoeboid; Small; Medium size;

compact; numerous numerous compact; few compact; few
chromatin masses; chromatin masses; chromatin chromatin masses;
coarse pigments; fine pigments masses; coarse pigments
rarely seen in coarse
peripheral blood pigments

Gametoc Crescent shaped, Spherical; compact Similar to P. Like P. vivax, but

yte larger and slender; vivax, but smaller
central chromatin smaller and
less
numerous
Study the permanent mounts, note your observations and identify the different stages:
References:

 Cappucino J and Sherman N. (2010). Microbiology: A Laboratory Manual. 9th

edition.Pearson Education Limited

 Willey JM, Sherwood LM, and Woolverton CJ. (2013) Prescott, Harley and Klein’s
Microbiology. 9th edition. McGraw Hill Higher Education

 http://www.uwyo.edu/molb2210_lab/

 https://science.umd.edu/classroom/bsci424/LabMaterialsMethods/BrothTubeMIC.htm

 https://www.cdc.gov/

 Basic Malaria Microscopy (part I and II) (WHO; 1991)