EXPERIMENT NO 1

STUDY OF PATHOGENIC BACTERIA

AIM: To study the pathogenic bacteria (E. coli, Salmonella, Pseudomonas, Staphylococcus
Bacillus) based on the cultural, morphological, and biochemical characteristics such as IMViC,
TSI, nitrate reduction, urease production, catalase and KOH solubility tests.

PRINCIPLE: The principle behind studying pathogenic bacteria using cultural, morphological,
and biochemical tests is to identify and characterize different bacterial species based on their
growth patterns, cellular morphology, and metabolic activities. The chosen tests provide
information about the bacteria's ability to ferment specific sugars, produce certain enzymes,
and exhibit other biochemical reactions. Here's the principle for each category of tests:

Cultural Characteristics: Different bacterial species have unique nutritional requirements and
environmental preferences. Cultural characteristics involve the cultivation of bacteria on
specific media under controlled conditions. Growth patterns, colony morphology, and
characteristics such as color, size, shape, and texture are observed.

Morphological Characteristics: Microscopic examination of bacterial cells provides

information about their size, shape, arrangement, and Gram-staining properties.

Biochemical Tests:

IMViC Tests: (Indole, Methyl red, Vogues Proskauer and Citrate)

Indole production: bacteria produce indole by breaking down tryptophan.

Methyl Red test: measures the ability of bacteria to perform mixed-acid fermentation.

Voges-Proskauer test: detects the production of acetoin, a precursor to 2,3-butanediol.

Citrate Agar: detects the ability of the bacteria to use sodium citrate with the production of
the enzyme citrate permease.

TSI (Triple Sugar Iron) Agar: tests for the ability of bacteria to ferment sugars, and produce
gas, and hydrogen sulfide.

Nitrate reduction test: determines the ability of bacteria to reduce nitrate to nitrite or other
nitrogenous compounds.

Urease production test: detects the enzyme urease, which hydrolyzes urea to produce
ammonia.

Catalase Test: detects the presence of catalase, an enzyme that catalyzes the breakdown of
hydrogen peroxide to water and oxygen.
KOH solubility test: an alternative for Gram staining, detects Gram’s nature of the bacteria
based on the bacteria to produce a viscous material on mixing with KOH.

The presence or absence of certain characteristics may provide insights into the pathogenic
potential of the bacteria. For example, the ability to ferment certain sugars or produce specific
enzymes might be associated with pathogenicity.

By applying these principles, the experiment aims to provide a detailed profile of each
bacterial species, enabling the differentiation and identification of pathogenic bacteria based
on their unique characteristics.

MATERIALS REQUIRED

Test Cultures: E. coli, Salmonella, Pseudomonas, Staphylococcus and Bacillus

Media used: Nutrient Agar, Tryptophan broth, MRVP broth, Citrate Agar slant, TSI Agar,
Nitrate broth, Urea agar slant.
Reagents: Kovac’s reagent, MRVP reagent, Sulphanilic acid, α naphthalamine, H2O2, KOH,
Grams Iodine, Acetone Alcohol,
Stains used: Crystal violet, safranine
Others: Petri dishes, test tubes, slides, glass rod, droppers, inoculation loop, inoculation
needle, Bunsen burner, etc.
Equipment: Autoclave, Incubator, Laminar air flow, Hot air oven, Microscope

PROCEDURE

1) Cultural Characteristics: Inoculate each bacterial strain (E. coli, Salmonella, Pseudomonas,
Staphylococcus, Bacillus) onto appropriate solid media (such as nutrient agar or specific
selective media for each). Incubate cultures at 37°C for 24hrs to observe growth patterns
and colony characteristics.

2) Morphological Characteristics: Perform Gram staining to observe the bacterial cell

morphology (Gram-positive or Gram-negative). Use a microscope to observe cell shape
(cocci, bacilli, spirilla) and arrangement.

3) Biochemical Tests:
IMViC Tests:
• Indole Production: Inoculate bacteria in tryptone broth and test for indole production
using Kovac's reagent. Observe for Cherry red ring on the addition of Kovac’s reagent.
• Methyl Red Test: Inoculate bacteria in MR-VP broth and test for the presence of acid
by adding methyl red. Observe for colour change to red on the addition of MR reagent.
• Voges-Proskauer Test: Inoculate bacteria in MR-VP broth and test for the production
of acetoin (positive for certain enteric bacteria). Observe for colour change to pink on
the addition of VP reagent
 • Citrate Test: Inoculate bacteria onto citrate agar slant and observe for colour change
in the slant from green to blue.

TSI (Triple Sugar Iron) Agar: Inoculate bacteria onto the TSI agar slant and observe for gas
production, hydrogen sulfide production, and the fermentation of sugars.

Nitrate Reduction Test: Inoculate bacteria in nitrate broth and check for nitrate reduction.
Observe for colour change to red on the addition of sulphanilic acid and alpha
naphthylamine reagent. Perform additional tests (e.g., adding zinc) to confirm the results.

Urease Production Test: Inoculate bacteria in urea slant and observe for the production
of ammonia, slant colour changes to pink.

Catalase Test: Test for the presence of catalase by adding hydrogen peroxide to the
bacterial colony. Development of effervescence/bubbling indicates the test positive.

KOH solubility: Test for Gram’s nature of the bacteria by adding KOH to the bacterial
colony. Gram-positive bacteria do not produce viscoid material but Gram-negative
bacteria produce a viscoid material due to high lipid content.

OBSERVATION (RHS): Use separate pages to write observations for each organism.

E. coli

Culture Characteristics: smooth moist non pigmented colonies on nutrient agar plates.

Gram’s Staining: Reddish pink rod shaped

Indole test: Production of Cherry red ring on addition of Kovac’s reagent.

MR test: Production of Red colour on addition of MR reagent.

VP test: No colour change in the broth on addition of VP reagent.

Citrate utilization: No growth of the bacteria and no colour change from green to blue

TSI: A/A -yellow slant/yellow butt with the production of gas, indicates glucose, lactose and
sucrose is fermented.

Nitrate reduction test: Appearance of red colour on addition of sulfanilic acid and α
naphthylamine reagent.

Urease test: No colour change in the slant

Catalase test: Immediate bubbling appears on addition of hydrogen peroxide

KOH Solubility test: viscoid material appears on mixing with KOH

Stick photos of the Normal colony in Nutrient Agar plate, Gram s Staining picture, and
Biochemical tests on the LHS for each organism.

Salmonella

Culture Characteristics: Non lactose fermenter, no pink coloured colonies in MacConkey

Agar, Smooth colonies often with a black centre on SS Agar.

Gram’s Staining: Reddish pink rod shaped

Indole test: No appearance of Cherry red ring on addition of Kovac’s reagent.

MR test: Production of red colour on addition of MR reagent.

VP test: No colour change in the broth on addition of VP reagent.

Citrate utilization: No growth of the bacteria and no colour change from green to blue

TSI: K/A -Red slant/yellow butt with the production of gas, indicates glucose is fermented.
Blackening of the medium indicating sulfur reduction.

Nitrate reduction test: Appearance of red colour on addition of sulfanilic acid and α
naphthylamine reagent.

Urease test: No colour change in the slant

Catalase test: Immediate bubbling appears on addition of hydrogen peroxide

KOH Solubility test: viscoid material appears on mixing with KOH

Pseudomonas

Culture Characteristics: often produces a characteristic fluorescent pigment.

Gram’s Staining: Reddish pink rod shaped

Indole test: No appearance of Cherry red ring on addition of Kovac’s reagent.

MR test: No production of red colour on addition of MR reagent.

VP test: No colour change in the broth on addition of VP reagent.

Citrate utilization: Appearance of growth and colour of the slant changes from green to blue

TSI: K/K No colour change indicating that the organism does not produce acid and gas and
sulfur reduction is negative.
Nitrate reduction test: No red colour on addition of sulfanilic acid and α naphthylamine
reagent.

Urease test: Colour change in the slant to pink

Catalase test: Immediate bubbling appears on addition of hydrogen peroxide

KOH Solubility test: viscoid material appears on mixing with KOH

Staphylococcus

Culture Characteristics: often forms creamy or opaque colonies.

Gram’s Staining: Bluish purple round shaped bacteria found in clusters

Indole test: No appearance of Cherry red ring on addition of Kovac’s reagent.

MR test: Production of red colour on addition of MR reagent.

VP test: No colour change in the broth on addition of VP reagent.

Citrate utilization: Appearance of Growth and colour changes from green to blue.

TSI: K/A colour change indicating that the organism ferments only glucose and gas and
sulfur reduction is also negative.

Nitrate reduction test: No red colour on addition of sulfanilic acid and α naphthylamine
reagent.

Urease test: Colour change in the slant to pink

Catalase test: Immediate bubbling appears on addition of hydrogen peroxide

KOH Solubility test: viscoid material does not appear on mixing with KOH

Bacillus

Culture Characteristics: often forms large, flat, spreading, rhizoid like colonies.

Gram’s Staining: Purplish blue rod shaped

Indole test: No appearance of Cherry red ring on addition of Kovac’s reagent.

MR test: Production of red colour on addition of MR reagent.

VP test: Colour change to rose indole in the broth on addition of VP reagent.

Citrate utilization: Appearance of growth and colour of the slant changes from green to blue
TSI: A/K No colour change indicating that the organism ferments only glucose and gas and
sulfur reduction is also negative.

Nitrate reduction test: Appearance of red colour on addition of sulfanilic acid and α
naphthylamine reagent.

Urease test: No colour change in the slant

Catalase test: Immediate bubbling appears on addition of hydrogen peroxide

KOH Solubility test: viscoid material does not appear on mixing with KOH

OBSERVATION: Table to be drawn from the observation book (Use Sharp Pencil while
writing on the LHS).

Table: Morphological and Biochemical characteristics of the Pathogenic bacteria (LHS)

RESULTS

E. coli: Gram-negative bacilli, positive for --------------------, negative for ------------------------

tests.

Salmonella: Gram-negative bacilli, positive for --------------------, negative for --------------------

---- tests.

Pseudomonas: Gram-negative bacilli, positive for --------------------, negative for ----------------

-------- tests.

Staphylococcus: Gram-positive cocci, positive for --------------------, negative for -----------------

------- tests.

Bacillus: Gram-positive bacilli, spore-forming, positive for --------------------, negative for -----
------------------- tests.
EXPERIMENT NO 2

STUDY OF COMPOSITION AND USE OF DIFFERENTIAL MEDIA

AIM: To study the composition and use of important differential media such as Eosin
Methylene Blue (EMB) Agar, MacConkey Agar, Mannitol Salt Agar (MSA), Salmonella Shigella
(SS) Agar and Thiosulfate Citrate Bile Salt Agar (TCBS) for the identification of pathogenic
bacteria

PRINCIPLE: Differential media are types of culture media used in microbiology to distinguish
between different microorganisms based on their growth characteristics and metabolic
properties. These media serve a specific purpose in identifying pathogenic bacteria.
Differential media often possess selective components that inhibit the growth of certain types
of microorganisms while allowing the growth of others. This selectivity is achieved through
the inclusion of specific inhibitors, such as antibiotics or high salt concentrations, that target
particular groups of bacteria or through indicator systems, which are substances that undergo
visible changes in response to the metabolic activities of bacteria. Differential media exploit
the metabolic capabilities of microorganisms to create observable differences. For example,
the ability to ferment specific sugars, produce certain enzymes, or tolerate particular chemical
compounds can result in distinctive growth patterns or color changes on the medium.

1. Eosin Methylene Blue Agar (EMB)

Composition:
Ingredients Gms / Litre

Peptone 10.000
Dipotassium hydrogen phosphate 2.000
Lactose 5.000
Saccharose (Sucrose) 5.000
Eosin - Y 0.400
Methylene blue 0.065
Agar 22.000
Final pH (at 25°C) 7.2±0.2

Preparation: Suspend 35.96 grams in 1000 ml purified/distilled water. Mix until the
suspension is uniform. Heat to boiling to dissolve the medium completely. Sterilize by
autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Use: EMB Agar is selective for Gram-negative bacteria and differentiates between
lactose and non-lactose fermenters. Lactose fermenters produce colonies with a
metallic green sheen due to acid production, while non fermenters appear colorless.
 The coliforms produce purplish black colonies due to taking up of methylene blue-
eosin dye complex when the pH drops. The dye complex is absorbed into the colony.
Nonfermenters probably raise the pH of the surrounding medium by oxidative
deamination of protein, which solubilizes the methylene blue-eosin complex resulting
in colorless colonies.

2. MacConkey Agar
Composition:
Ingredients Gms / Litre
Gelatin peptone 17.000
HMC peptone 3.000
Lactose monohydrate 10.000
Sodium chloride 5.000
Bile salts 1.500
Neutral red 0.030
Crystal violet 0.001
Agar 22.000
pH after sterilization (at 25°C) 7.1±0.2

Preparation: Suspend 49.53 grams (the equivalent weight of dehydrated medium per
liter) in 1000 ml purified/distilled water. Boil for 1 minute with constant stirring.
Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes or as per the
validated cycle

Use: McConkey agar is both selective and differential. It inhibits the growth of Gram-
positive bacteria and differentiates between lactose and non-lactose fermenters.
Lactose fermenters produce pink colonies due to acid production, while non-
fermenters appear colorless. MacConkey Agar and Broth have been recommended for
use in the microbiological examination of foodstuffs and direct plating/inoculation of
water samples for coliform counts. This medium is also accepted by the Standard
Methods for the Examination of Milk and Dairy Products. It is recommended in
pharmaceutical preparations The red colour is due to the production of acid from
lactose, absorption of neutral red, and a subsequent color change of the dye when the
pH of medium falls below 6.8. Lactose non-fermenting strains, such as Shigella and
Salmonella are colourless and transparent and typically do not alter appearance of the
medium
3. Mannitol Salt Agar
Composition:
Ingredients Gms / Litre
Peptone 5.000
Tryptone 5.000
HM Peptone B 1.000
Sodium chloride 75.000
D-Mannitol 10.000
Phenol red 0.025
Agar 22.000
pH after sterilization (at 25°C) 7.4±0.2

Preparation:

Use: Mannitol salt agar is selective for Staphylococcus species, particularly

Staphylococcus aureus. It differentiates between mannitol fermenters (yellow
colonies) and non-fermenters (pink or red colonies). The high salt concentration
inhibits the growth of many other bacteria. Staphylococci have the unique ability to
grow on a high salt-containing media. Many other bacteria except Staphylococci are
inhibited by 7.5% sodium chloride. Mannitol is the fermentable carbohydrate
fermentation that leads to acid production, detected by the phenol red indicator. S.
aureus ferments mannitol and produces yellow-colored colonies surrounded by
yellow zones. Coagulase-negative strains of S. aureus are usually mannitol non-
fermenters and therefore produce pink to red colonies surrounded by red-purple
zones.

4. Samonella Shigella (SS) Agar

Composition:
Ingredients Gms / Litre
Peptone 5.000
HM peptone B 5.000
Lactose 10.000
Bile salts mixture 8.500
Sodium citrate 10.000
Sodium thiosulphate 8.500
Ferric citrate 1.000
Brilliant green 0.00033
Neutral red 0.025
Agar 22.000
Final pH (at 25°C) 7.0±0.2
 Preparation: Suspend 63.02 grams in 1000 ml purified /distilled water. Boil with
frequent agitation to dissolve the medium completely. DO NOT AUTOCLAVE OR
OVERHEAT.

Use: SS Agar medium is recommended as a differential and selective medium for the
isolation of Salmonella and Shigella species from pathological specimens and
suspected foodstuffs. SS Agar is a moderately selective medium in which gram-
positive bacteria and coliforms are inhibited by bile salts, brilliant green, and sodium
citrate. Sodium thiosulphate is reduced by certain species of enteric organisms to
sulfite and H2S gas and this reductive enzyme process is attributed to thiosulphate
reductase. Production of H2S gas is detected as an insoluble black precipitate of
ferrous sulfide, formed upon reaction of H2S with ferric ions or ferric citrate, indicated
in the center of the colonies. Growth of Salmonella species appears as colorless
colonies with black centers resulting from H2S production. Shigella species also grow
as colorless colonies which do not produce H2S.

5. Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS)

Composition:
Ingredients Gms / Litre
Yeast extract 5.000
Peptic digest of animal tissue 10.000
Sodium citrate 10.000
Sodium thiosulphate 10.000
Sodium cholate 3.000
Oxgall 5.000
Sucrose 20.000
Sodium chloride 10.000
Ferric citrate 1.000
Bromo thymol blue 0.040
Thymol blue 0.040
Agar 22.000
Final pH (at 25°C) 8.6±0.2

Preparation: Suspend 89.08 grams in 1000 ml distilled water. Heat to boiling to

dissolve the medium completely. DO NOT AUTOCLAVE.

Use: TCBS agar is selective for Vibrio species, particularly Vibrio cholerae. It
differentiates between sucrose fermenters (yellow colonies) and non-fermenters
(green colonies). This medium is commonly used for the isolation and identification of
 Vibrio cholerae. Strains of Vibrio cholerae produce yellow colonies on TCBS Agar
because of the fermentation of sucrose. Vibrio alginolyticus also produces yellow
colonies. Vibrio parahaemolyticus is a sucrose non-fermenting organism and produces
blue-green colonies, as Vibrio vulnificu

MATERIALS REQUIRED

Ingredients for media preparation – EMB, MacConkey Agar, MSA, SS Agar and TCBS Agar.
Test organisms and sample – E. coli, Salmonella, Staphylococcus, Marine water sample.
Glassware: conical flask, Petridishes, measuring jar,
Equipment: Autoclave, waterbath, weighing balance, Incubator, laminar air flow.

PROCEDURE
• Weigh the ingredients needed for various media preparation.
• Autoclave, EMB, MacConkey and MSA media at 121°C, 15lbs for 20 minutes.
• Do not Autoclave SS and TCBS agar
• Pour the media into sterile Petri dishes and allow it to solidify.
• Streak the test bacteria to the plates by quadrant streaking and zig-zag method
• Keep the plates for incubation a 37°C for 24 hrs.
• Record the observations and report the results.

OBSERVATION:
EMB Agar: Metallic green sheen colonies by E. coli were observed in the EMB plates.
MacConkey’s Agar: Lactose fermenters (E. coli) showed pink color colonies while the non-
lactose fermenters (Salmonella) were colorless.
MSA Agar: Pathogenic Staphylococcus (S. aureus) produced yellow colonies whereas,
nonpathogenic Staphylococcus (S. epidermidis) produced pink-colored colonies.
SS agar: Black-centered smooth colonies by Salmonella were observed.
TCBS Agar: Yellow-coloured colonies by Vibrio cholerae, as well as green colonies by Vibrio
parahaemolyticus, were observed.

RESULTS: Differential media play a crucial role in isolating and identifying pathogenic bacteria
based on their metabolic capabilities and other distinctive characteristics.
EXPERIMENT NO: 3.

ANTIBIOTIC SENSITIVITY TEST-KIRBY BAUER METHOD

AIM: To determine bacterial sensitivity to a given antibiotic by paper disc diffusion method.

PRINCIPLE: Certain bacteria can display resistance to one or more antibiotics. Determining
bacterial antibiotic resistance – whether a bacterium can survive in the presence of an
antibiotic - is a critically important part of the management of infectious diseases in patients.
The Kirby-Bauer (K-B) disk diffusion test is the most common method for antibiotic
resistance/susceptibility testing. The results of such testing help physicians in choosing which
antibiotics to use when treating a sick patient. The Kirby-Bauer (K-B) test utilizes small filter
disks impregnated with a known concentration of antibiotic. The disks are placed on a
Mueller-Hinton agar plate that is inoculated with the test microorganism. Upon incubation,
antibiotic diffuses from the disk into the surrounding agar. If susceptible to the antibiotic, the
test organism will be unable to grow in the area immediately surrounding the disk, displaying
a zone of inhibition. The size of this zone is dependent on a number of factors, including the
sensitivity of the microbe to the antibiotic, the rate of diffusion of the antibiotic through the
agar, and the depth of the agar. Microorganisms that are resistant to an antibiotic will not
show a zone of inhibition (growing right up to the disk itself) or display a relatively small zone.

MATERIALS REQUIRED
Forceps, Sterile swabs, Two Mueller-Hinton agar plates, Antibiotic disks-(i) TWO different
antibiotics (ii) Antibiotic-free disks (BLANK)
Stock broth cultures of: - Escherichia coli (Gram negative) - Staphylococcus aureus (Gram
positive).

PROCEDURE:
● Label the agar plates with the usual 5 items (Name of the media, date, organism,
antibiotic disc and your name). Also mark, using dots, where you will put the antibiotic
disks and the BLANK (antibiotic-free) disk.
● Disks should be a minimum of 20 mm apart.
● Disks should not be placed near the edge of the plate
● Inoculate one plate with your first bacterium as follows:
● Using aseptic technique, wet a swab with the bacterial broth culture.
● Thoroughly swab the surface of the plate, making sure to cover the entire surface.
 ● Do NOT get more culture.
● Turn the plate approximately 60 degrees and
repeat the previous step (2nd swabbing).
● Do NOT get more culture.
● Repeat the previous step (3rd swabbing).
● Discard the swab in a bleach-containing
beaker.
● Place one antibiotic disk onto the surface of
the agar, using aseptic technique as follows:
Flame the tips of the forceps by passing over the
flame for 5-10 seconds. Cool the forceps by
waving them in the air for about 10 seconds.
● Carefully pick up your test disk with the forceps, and gently place it in the appropriate
spot on the agar surface,
● To ensure that the disk is flat on the agar, gently push it down with the forceps.
● Reheat the tips of the forceps as above to kill any bacteria.
● Repeat the procedure with the second antibiotic disk.
● Repeat the procedure with the BLANK disk.
● Repeat steps 1 – 6 on a new agar plate with your second bacterium.
● Wait until the surface of the plates has completely dried (it may help to leave the lid
slightly open for 3 - 5 minutes).
● Incubate both plates at 37°C for 18-24 hours.
● Observe both agar plates for zone of inhibition.
● If present, measure the diameter of the zone of inhibition in mm (see the figures below),
and record your results in the Table.
● If there is no zone present, record your result as 0 mm.
OBSERVATION:

Table: Antibacterial activity of Antibiotics by disc diffusion method

Sl.No. Antibiotic & Concentration Zone of inhibition (mm)

E. coli Staph aureus

R – resistant, I – intermediate, S - sensitive

RESULTS: E. coli is sensitive to ……………………….. and Staph is sensitive to………………….

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