The document describes methods for isolating and identifying microbes from various samples. It discusses taking soil, water, food, and other samples and performing serial dilutions to isolate colonies. It then explains how to conduct purity plating to obtain pure cultures, and identifies biochemical tests including gram staining, mannitol salt agar testing, and coagulase testing to identify microbes like Staphylococcus aureus. The goal is to isolate both beneficial and pathogenic microbes from the environment using microbiological techniques.

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Amjad Khan Afridi 25 /07/ 2016
1. Soil sample
2. Tap Water sample
3. Slag sample
4. Food sample
5. Milk sample
Etc……
Purpose:
To isolated antibiotic production microbes ( Bacteria or Fungi ) from
samples.
To isolated degradable microbes ( Bacteria or Fungi ) , to removed
contamination and to keep and clean the environment.
To isolated infective or pathogenic microbes ( Bacteria or Fungi ) from food
, water and milk samples.
To observed and to isolated the different microbes from the environment
that are present in different seasons ( summer , winter , spring , autumn ) .
SERIAL DILUTION METHOD
Serial dilution method is one of the most old and usable method which is use for
the isolation of bacterial colony.
In this method we take concern samples ( soil , slag, water, milk, food ) make its
dilution in the test tube.
We inoculated the sample from the diluted test tubes in the prepared Nutrient Agar
plates by using Pure Plat Methodand then incubated the cultured plates at 37 C
for 24 hours.
After 24 hours we absorb the cultured plates , the growth will appear on each
plates.

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Next we performed sub culturing method for the isolation of pure culture. After
isolation of pure culture we perform gram staining method and other Biochemical
test for the conformation and identification of bacterial species.
Materials:
1) Samples (Soil, Tap water, Food, Slag, Milk ) .. etc
2) Saline solution or Distilled water
3) Nutrient Agar
4) Beaker
5) Flask
6) Graduated cylinder
7) Test tubes
8) Test tubes stand
9) Media plates
10) Electric balance
11) Aluminum foil
12) Cotton
13) Micro peptide
14) Burner
15) Wire loop
16) Ethanol
17) Para film
18) Autoclave
19) Incubator
20) LFH
21) Marker
PROCEDURE:
1) Collected desire Samples (Soil, Tap water, Food, Slag, Mild) from the
environment
2) Take 8 test tubes and take 10 ml of saline in 1st test tube and take 9ml of
distilled water in each the remaining test tubes and then labeled each test
tubes.

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3) Take 1 gram of Samples (Soil, Food, Slag, Milk) and make a solution in
the 1st test tubes which having 10 ml of Saline solution. Which will called
Master test tube.
4) Distribute the sample solution from the 1st
test tube( master test tube) in the
remaining test tubes.
Take 1 mal of sample solution from 1st
test tube ( Master test tube) and put in the
2nd
test tube.
Take 1mal of sample solution from 2nd test tube and put in the 3rd test tube.
Take 1 mal of sample solution from 3rd test tube and put in the 4th
test tube.
Take 1 mal of sample solution from 4th test tube and put in the 5th
test tube.
Take 1 mal of sample solution from 5th
test tube and put in the 6th
test tube.
Take 1 mal of sample solution from 6th
test tube and put in the 7th
test tube.
Take 1 mal of sample solution from 7th
test tube and put in the 8th
test tube.
5) Prepared Nutrient Agar media ( NA ) .
6) For the purring the samples in the media plates we use Purring Plate
Method .
7) By using micro peptide We take .5 ml of sample solution from the desire
test tubes and purring on the media plates by Purring Plate Method.
8) First we purring the sample solution in the test tubes and then we purring the
Nutrient Agar in the plates. And labeled the plats also.
9) Allow the plates to solidified and then we replaced the inoculated media
plates in the incubator at 37 C for 24 hours.
10) After 24 hours we absorb the cultured plates, the growth of microbes
will be appeared on the media plates.
11) Next we perform sub culturing for the isolation of pure culture.
12) Again we prepare new fresh Nutrient Agar, purring the media in the
plates. Allow the plates to solidified and then we pick up a single colonies
from each growth cultured plates and inoculated on fresh Nutrient Agar
plates by using striking method.

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13) After striking replaced the inoculated plates in the incubator at 37 C
for 24 hours.
14) After 24 hours a pure, clear and visible colonies will appeared on
each plates.
15) Next we use these pure culture for gram staining as well as for
biochemical tests for the conformation and identification of the isolated
bacterial colonies.
Note:
As the same method we can isolated microbes from slag , food and milk samples.
We use the same method and the same procedurefor the isolation of microbes
from these samples.

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The isolation of microbes from the open environment ( Air ) is very easy and simple.
Materials:
1. Nutrient Agar
2. Beaker
3. Flask
4. Electric balance
5. Aluminum foil
6. Cotton
7. Burner
8. Ethanol
9. Para film
10. Autoclave
11. Incubator
12. LFH
13. Gloves
14. Mask
Procedure:
 Prepared Nutrient Agar and purring it in the media plates. Removed the
upper lid from the media plate and keep the plates for 10 to 15 minutes in
the open area. After 15 minutes covered the media plate and replaced the
plate in the incubator for 24 hours at 37 C.
 After 24 hours absorb the plate , the growth of microbes will appeared .
 For getting the pure culture we perform sub culturing method .
 Again we prepared new fresh Nutrient Agar media and purred in the media
plate and allow it to solidified.
 After solidification we pick up a single colony from the growth cultured
plate by using wire loop and strike on fresh media plate.

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 After striking we replaced the inoculated plates in the incubator at 37 C for
24 hours.
 After 24 hours a clear, fresh and pure growth will appeared on the cultured
plate.
 Next we use this pure culture for gram staining as well as for biochemical
tests for the conformation and identification of the isolated bacterial
colonies.
Staphylococcus aureus is a type of bacteria. It stains Gram positive and is non-
moving small round shaped or non-motile cocci.
It is found in grape-like (staphylo-) clusters. This is why it is called
Staphylococcus.
Staphylococcus aureus belongs to the family Staphylococcaceae
S. aureuscan be readily transmitted from one species to another. This includes
transmission between humans and animals.
Staphylococcus is one of the five most common causes of infections after injury or
surgery.
It is abbreviated to “S. aureus” or “Staphaureus” in medical literature.
S. aureuswas discovered in Aberdeen, Scotland in 1880 by the surgeon Sir
Alexander Ogstonin pus from surgical abscesses.
Of the variety of manifestations S. aureus may cause:
 Minor skin infections, such as pimples, impetigo etc.
 It may cause boils (furuncles), cellulitis folliculitis, carbuncles
 It is the cause of scalded skin syndrome and abscesses
 It may lead to lung infections or pneumonia
 Brain infections or meningitis
 Bone infections or osteomyelitis

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 Heart infections or endocarditis
 Generalized life threatening blood infections or Toxic shocksyndrome
(TSS), bacteremia and septicaemia.
Manitol Salt Agar is a pink like color media which is used for the identification of
S.aureus. MSAdifferentiating S.aureusfrom other Staphylococcus aureus
species.
S.aureuschanged MSA color from pink into yellow by utilizing and fermentation
the MSA, and by releasing acid which reduced its pH.
If the color of MSA is changed from pink into yellow , its mean S.aureus
fermented the MSA and produced the acid.
Materials:
1) Manitol Salt Agar ( MSA )
2) Samples ( S.aureus)
3) Beaker
4) Flask
5) Electric balance
6) Aluminum foil
7) Cotton
8) Burner
9) Ethanol
10) Para film
11) Autoclave
12) Incubator
13) LFH
14) Gloves
15) Mask

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Procedure:
Prepared Manitol Salt Agar (MS) in a single media plate . Purring the media plate
by MSA and allow it to solidified it.
After solidification sterile the wire loop and make dissected the MSA media in the
plate 1 and 2.
Again sterile the wire loop and pick a colony (S.aureus)from the broth media and
strike on the 1st
section of the media plate .
Next again sterile the wire loop and pick other species colony from broth media
and strike on the 2nd
section of the MSA media plate.
Covered the inoculated MSA plate by Para film and replace in the incubator for 24
hours at 37 C.
After 24 hours absorb the cultured plate. The 1st
section where we inoculated the
S.aureuswill changed its color from pink into yellow , while the 2nd
section color
will not changed.
COAGULASE TEST
Staphylococcus aureus is known to produce coagulase, which can clot plasma
into gel in tube or agglutinate cocci in slide.
This test is useful in differentiating S.aureusfrom other coagulase-negative
staphylococci. Most strains of S.aureusproducetwo types of coagulase,
free coagulaseand bound coagulase.
The enzyme coagulaseis demonstrated in vitro by two methods
a. The Slide coagulase test
b. The Tube coagulase test
a) The Slide coagulase test

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Principle:
This method measures bound coagulase. The bound coagulase is also known as
clumping factor. It cross-links the α and β chain of fibrinogen in plasma to form
fibrin clot that deposits on the cell wall. As a result, individual coccus stick to each
other and clumping is observed.
Procedure:
1. Divide the slide into two sections with grease pencil. One should be
labeled as “test” and the other as “control
2. 2 Place a small drop of distilled water on each area
3. Emulsify one or two colonies of Staphylococcus on blood agar plate on
each drop to make a smooth suspension
4. The test suspension is treated with a drop of citrated plasma and mixed
well with a needle
5. Do not put anything in the other drop that serves as control. The control
suspension serves to rule out false positivity due to auto agglutination
6. Clumping of cocci within 5-10 seconds is taken as positive.
7. Some strains of S.aureus may not produce bound coagulase, and such
strains must be identified by tube coagulase test
b) The Tube Coagulase Test
Principle:

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This method helps to measure free coagulase. The free coagulase
secreted by S.aureus reacts with coagulase reacting factor (CRF) in
plasma to form a complex, which is thrombin. This converts fibrinogen
to fibrin resulting in clotting of plasma.
Procedure:
1. Three test tubes are taken and labeled “test”, “negative control” and “positive
control”.
2. Each tube is filled with 1 ml of 1 in 10 diluted rabbit plasma.
3. To the tube labeled test, 0.2 ml of overnight broth culture of test
4. bacteria is added.
5. To the tube labeled positive control, 0.2 ml of overnight broth culture of known
S.aureus is added
6. To the tube labeled negative control, 0.2ml of sterile broth is added.
7. All the tubes are incubated at 37oC and observe the suspensions at half hourly
intervals for a period of four hours.
8. Positive result is indicated by gelling of the plasma, which remains in place even
after inverting the tube.
9. If the test remains negative until four hours at 37oC, the tube is kept at room
temperature for overnight incubation.
Demonstrated By MadamAzra
Prepared By Amjad Khan Afridi
Date: 25 /07/2016