Microbiology Assignment

“Isolation and
Identification of the
causative bacteria:
Nasal Disorder”

Submitted by: Kapil Rathi

Enroll. No: V/18/312

Guided by: Dr, Pawde ma’am

Step – 1: Collection of Sample- Suspected for nasal
disorder

Details of animal:

1. Breed: Khillar
2. Sex: Male
3. Age: 4yrs

Symptoms:

1. Nasal discharge
2. Fever
3. Shortness of breath

Method of collection: Nasal swab

 Ideally, a sterile, factory made nasal swab should be used.

 But, if not available, makeshift nasal swabs, made using a thin skewer/stick with sterile
cotton bud/ball at end, may be used
 The bud end is rolled in the inner exposed part of the nostrils, so collecting adequate
amount of nasal discharge
Step-2: Inoculation in Nutrient Broth

1. Preparation of Nutrient broth

Nutrient broth is used for the very initial growth of bacteria, in the samples/specimens collected,
to enable further isolation.

Composition of nutrient broth:

Ingredients gm/litre
Beef extract 1

Sodium chloride 0.5

Peptone 1

Distilled water 100ml

Preparation:

- Add 1.3g of nutrient broth powder in 100ml of distilled water.

- Dissolve it completely.
- Sterilize by autoclaving at 121C for 15mins

2. Inoculation:

- Take nutrient broth in test-tube.

- Now, put the nasal swab in it such that the cotton part remains wholly submerged in the
broth.
- Close the test-tube with a sterilized cotton plug or cork.
3. Incubation:

- Place the testube in an incubator overnight at 37C.

4. Observation:

- Turbidity is seen in the test-tube due to the growth of bacteria.

This is is the test-tube of nutrient

broth, inoculated within which is
the nasal swab for the initial
bacterial multiplication.
Step-3: Streaking on Nutrient Agar

1. Preparation of Nutrient agar media

- Nutrient agar is primarily used as a general (solid) for the growth of organisms with
colony formation.
- Nutrient broth didn’t provide with colonies and hence, not that immediate useful for
streaking.

Composition of NA

Ingredients gm/l

Peptone 5

Sodium chloride 5

HM peptone B 1.5 The apparatus mainly needed for streaking:

Inoculation loop, flame source and sterilized
Yeast extract 1.5
media/agar plate viz. Nutrient agar here.
Agar 1.5

Preparation:

- Suspend 28 gms of Nutrient agar powder in 100ml distilled water.

- Heat to dissolve it completely.
- Sterilize by autoclaving it at 15lbs pressure for 15mins.
- Cool it down and then pour it on the petridishes.

2. Streaking on nutrient agar plates:

- Take a nutrient agar plate, sterilize the inoculation loop.
- Dip it in the nutrient broth and then carefully streak it on the nutrient agar plate.

3. Incubation:

- Incubate the streaked NA plate for 12-14 hours.

4. Observation:

- After the incubation period, pearl white colonies were observed.

5. Precautions:

- Maintain sterile conditions at all times.

- The streaking should be done in front of a flame only.
- Don’t overincubate the plates as it increases the chances of contamination and also,
getting a small colony gets harder.

This is a clear case of

contamination of slide due to If properly streaked and
overincubation and not incubated, such pearl white
maintaining sterile colonies are obtained
environment
Step-4: Gram Gram staining of culture,
Staining
showing pink coloured rods

Prepare a smear on a clean, glass slide and stain it

using gram stains.

- Pour primary stain, Crystal violet, allow reaction for 1min and then wash off.
- Then pour Grams iodine, keep it for 1min and wash off.
 - Now, Pour a few drops of decolouriser-alcohol, keep for 30secs and wash off.
- Pour counterstain, safranin, allow reaction for 1min and dry the slide
- Air dry the slide
- Observe under 100x oil immersion

Observation
- Pink coloured rods were visualized, indicating Gram negative bacilli.

Step-5: Streaking on Blood agar

1. Preparation
Composition

Ingredients g/l

Peptone 10.0
Sodium chloride 5.0
Beef extract 10.0
Agar powder 15.0
Water 1000ml
Sheep blood, 50.0ml
(defibrinated)

- Add all the components (40gm) except sheep blood, to distilled/deionized water and
bring volume to 950.0 ml.
- Mix thoroughly.
- Heat with frequent agitation and boil for 1 min to The white, opaque, mucoid,
completely dissolve. hemolytic colonies observed on
the blood agar
- Autoclave for 15mins at 15psi pressure at 121C.
- Cool to 45-50C.
- Aseptically add 50.0ml of sterile, defibrinated sheep blood.
- Mx throroughly and pour into petri dishes.

2. Streaking:

- Take a Blood agar plate.

- Pick an isolated colony from the nutrient agar with the help of sterilized inoculation loop.
- Streak it on the Blood agar plate in a sterilized medium.

3. Incubation:

- Keep the agar plate for incubation, overnight.

 4. Observation:

- Hemolytic,white coloured,mucoid colonies were observed.

5. String test

- Using a sterile inoculation loop, try to pick a

colony at random.
- Measure the length upto which the colony
stretches, without breaking the string.

Observation: - The colon stretches upto more than

5mm, hence, test positive for hypermucoviscosity.

- The colonies obtained on Blood agar

are of a virulent strain.

String test of obtained colonies,

testing them positive for
hypermucoviscosity
Step-5: Streaking on MacConkey Agar:

6. Preparation
Composition

Ingredients g/l

Peptone 2
Sodium taurocholrate 0.5
Sodium chloride 0.5
Lactose 1
Agar powder 2
Water 100

- Suspend 4.953gm of dehydrated medium in 100ml of

distilled water.
- Heat to dissolve completely.
- Sterilize by autoclaving at 121C for 15mins.
- Cool it down and pour in a petri dish while still liquid.

7. Streaking:

- Take a MacConkey agar plate. Such pink colonies were

obtained on incubation of the
- Pick an isolated colony from the blood agar plate with
streaked MacConkey agar plate,
the help of sterilized inoculation loop. indicating lactose-fermenting
- Streak it on the MacConkey agar plate in a sterilized bacteria
medium.

8. Incubation:

- Keep the agar plate for incubation, overnight

9. Observation:

- Pink coloured colonies were observed, indicating the lactose-fermenting nature of the
bacteria.
Step-6: Streaking on EMB agar

1. Preparation:

Composition

Ingredients gm/l

Peptone 10.0
Di-pottasium hydrogenphosphate 2.0
Lactose 5.0
Sucrose 5.0
Eosin Y 0.4
Methylene blue 0.07
Agar 13.5
Distilled water 1000ml
EMB agar plate

- Suspend 36gm of dehydrated medium in 1000ml distilled water.

- Heat to dissolve medium completely.
- Dispense and sterilize by autoclaving at 15lbs pressure for 15mins at 121C.
- After cooling, pour into sterile petri plates at 25C.

2. Streaking:

- Take an EMB agar plate and using a sterilized inoculation loop, pick an isolated colony
from the MacConkey agar plate and streak it on the EMB agar plate.
3. Incubation:

- Incubate the streaked EMB agar plate overnight, at 37C.

4. Observation:

- Large, mucoid, pink with purple centres colonies, without metallic sheen are observed,
indicating Klebsiella species.
Step-7: Streaking on Coliform ChromoSelect agar

1. Preparation:

Composition

Ingredients gm/l

Peptone, special 3.0

phosphate 3.0
Potassium dihydrogen phosphate 1.7
NaCl 5.0
Sodium laryl sulfate 0.1
Sodium pyruvate 1.0
Tryptophan 1.0
Chromogenic mixture 0.2 Coliform Chromogenic Agar

Agar 12.0
Distilled water 1000ml

- Suspend 27gm of dehydrated medium in 1000ml distilled water.

- Heat to boiling, dissolve medium completely.
- Sterilize by autoclaving at 15lbs pressure for 15mins at 121C.
- After cooling, pour into sterile petri plates at 25C.

2. Streaking:
- Take an Klebsiella Coliform ChromoSelect agar ( KCCS) plate and using a sterilized
inoculation loop, pick an isolated colony from the EMB agar plate and streak it on the
KCCS agar plate.

3. Incubation:

- Incubate the streaked KCCS agar plate overnight, at 37C.

4. Observation:

- Large, mucoid, pink colonies, without metallic sheen are observed, indicating Klebsiella
species.
Conclusion:

Considering all the the sample collected tests positive for

Klebsiella pneumoniea.