Identification of gram positive and gram negative bacteria

Identification of gram positive and gram negative bacteria

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Amjad Khan Afridi 10
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August, 2016
Identification of gram positive and gram negative bacteria
OBJECTIVE:
 To know the isolated bacterial cultural either its gram negative or gram positive.
For the identification of gram positive and gram negative bacteria we follow the main two steps.
A. Gram Staining
B. Biochemical tests
(A) Gram Staining:
It’s a monumental technique.
Gram Staining is a common technique used to differentiate two large groups of
bacteria based on their cell wall constituents. The gram staining procedure
distinguishes between Gram Positive and Gram Negative groups of bacteria by
coloring their cells red or violet.
Gram Positive and Gram Negative bacteria can be identify by their respective
color. The basis of this difference is staining is the structure of the cell wall . Both
Gram Positive and Gram Negative bacteria cell walls contains a layer of
peptidoglycan. Gram Positive has thick peptidoglycan while Gram Negative has
thin peptidoglycan.
DIFFERENCE IN BETWEEN GRAM POSITIVE AND GRAM NEGATIVE BACTERIA CELLS.
Names Gram Positive Gram Negative
Peptidoglycan Thick Thin
Teichoic Acid Present Absent
LPS ( Lipid Poly Saccharide) Absent Present
Proteins content Absent Present
Releasing Endospores No Yes
Thickness of cell wall 20 to 80 nm 10 nm
Out membrane Absent Present
Porins proteins Absent Present
Permeability of molecules More permeable Less permeable

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Amjad Khan Afridi 10
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MATERIALS:
1. 24 hours fresh bacterial cultural
2. Crystal violet
3. Iodine
4. De-colorizer ( Ethyl alcohol)
5. Safranin
6. Oil ( Immersion Oil )
7. Distilled water
8. Wire loop
9. Glass slides
10. Slides covers
11. Sprite lamp / Burner
12. Slides stray
13. Bibulous paper / absorbent paper
14. Beaker
15. Gloves
16. Mask
17. Permanent marker
18. Ethanol
19. Soap
PROCEDURE:
1. Take distilled water and dropped a single drop of distilled water on the
surface of the glass slide.
2. Serialized the wire loop and pick a colony from the growth medium and
mixed with drop on the glass slide and formed a smear and dried it.
3. After heat fixing, Next apply the chemicals on the smear slide.
4. Crystal Violet: Its also called primary dye. Its purple like color.
 Pass crystal Violet from the fixed smear and allow it for 1 minute.
 After I minute rains off the crystal violet with distilled water or tap
water.
 Its adjusted in the peptidoglycan of the bacterial cell wall.
 Gram negative bacteria take this basic dye due to their thin
peptidoglycan.

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5. Iodine: Also called moderate dye. It is red like color.
 Next iodine is passed from the smear for 1 minute, to fixed the
primary dye ( crystal Violet) in the cell wall of gram Negative
bacteria.
 Rains off the iodine after 1 minute by distilled water or tap water.
 With this both gram negative and gram positive bacteria will appear
violet or purple.
6. De-colorizer ( Ethyl alcohol) : It’s a color less dye.
 This is discoloring agent which remove purple color from some
species.
 Also remove lipids from the cell wall of gram positive bacteria if we
apply for more times.
 De-colorizer 95 % ( Ethyl alcohol, Alcohol ) is apply for a short
time ( 5sec to 10 sec).
 Rains off the De-colorizer from the slide with distilled water or tap
water.
7. Safranin:
 Also called secondary dye.
 After washing the De-colorizer , Safranin is apply for 45 sec to 1
minute.
 This secondary dye is fixed in the cell wall of gram positive bacteria.
 Fixed in the pores of gram positive bacteria.
 Rains off the safranin from the smear and allow the smear to dry.
8. Microscopy:
 After drying the slide,
 Covered the slide ,
 Dropped a single drop of immersion oil on the slide
 Adjust the slide under the microscope , adjust with 40 lens and
absorbed with 100 lens.
 100 lens is also called oil lens.
 It has same reflective index and same medium to glass slide.
 You will able to know either the smear is gram positive or gram
negative.
9. Result:

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 Gram positive bacteria will appear purple color
 Gram negative bacteria will appear pink color.
Precautions:
 24 hours fresh culture should be present.
 Wire loop should be sterilized.
 Do not touch your fingers , tongue, eyes, and face with chemicals. Because
these caused cancer.
 Gloves, mask, lab coat should be used.
 All chemicals should be used as proper sequence (step wise).
 Do not use each chemical (dye) more than their limit time.
 Take slides with safety and hold it tightly , either do not cut your fingers.
 Be careful when using microscope.
 Discard each slides properly after finishing your procedure.
 Wash your hands with soap or ethanol before leaving the laboratory.

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Amjad Khan Afridi 10
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August, 2016
(B) BIOCHEMICAL TESTS
For the identification of gram positive and gram negative bacteria we use the
following biochemical tests.
1. Catalase Test
2. Coagulase Test
3. Oxidase Test
4. Indole Test
5. Citrate Test
6. TSI ( Triple Sugar Iron) Test
7. Urease Test
(1) CATALASE TEST
S.aureus are catalase positive . it degraded Hydrogen peroxide in to water and
oxygen , during which production of bubbles . These bubbles indicates positives
reaction , while absence of bubbles indicates negative reaction.
catalase is biochemical test which is use for the identification of Gram positive
bacterial species either these produced catalase enzyme or not. Catalase enzyme
detoxifies hydrogen peroxide by breaking down into water and oxygen.
Staphylocococcus spp. and Micrococcus spp. are catalase positive.
The streptococcus and Enterococcus spp. are catalase negative.
Materials:
1. Fresh S.aureus bacterial cultured ( 24 hours fresh )
2. Glass slides
3. Wire loop or ( wood sticks )
4. Hydrogen peroxide 3%
5. Test Tubes
6. Test tube stand
7. Bunsen burner
8. Syringe
9. Glass slides

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Procedure:
The test can be done by two methods.
a. Slant method
b. Slide method
a) Slant Method:

1. Using a sterile technique, inoculate each experimental organism into its
appropriately labeled tube by means of a streak inoculation.
2. Incubate all cultures for 24-48 hours at 37˚C.
3. Allow three or four drops of the 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.

B) SLIDE METHOD:
Take a fresh glass slide and dropped 3% of Hydrogen peroxide by using syringe.
Next puck up a fresh bacterial ( S.aureus ) culture colony by using wire loop or
wooden sticks and mixed with drops on the slide . wait for few seconds , the
bubbles will produced .
If the bubbles are produced its mean the tested bacterial cultural is positive against
catalase test.

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(2) 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.aureus from other coagulase-negative staphylococci. Most strains of S.aureus
produce two types of coagulase, free coagulase and bound coagulase.
Coagulase is an enzyme that clots blood plasma. This test is performed on gram
positive bacteria. It’s used to identify the Coagulase positive species like
Staphylococcus aureus. Coagulase is virulent factor of s. aureus.
This test differentiations Staphylococcus aureus from other coagulase negative
Staphylococcus aureus species.
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.
The enzyme coagulase is demonstrated in vitro by two methods
a. The Slide coagulase test
b. The Tube coagulase test

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A) THE SLIDE COAGULASE TEST

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:
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.

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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 37˚C 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 37˚C, the tube is kept at room
temperature for overnight incubation.



(+) --- Delayed reaction
d --- 11- 89% of strains are positive

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Amjad Khan Afridi 10
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August, 2016
(3) OXIDASE TEST:
Oxidase Test is a quick biochemical Test which perform for the identification of
Gram Positive bacteria.
Oxidase test identifies bacteria which produced an enzyme called Cytochrome C
Oxidase. This enzyme help in electron transport chain, which carry electron from
donor molecule to oxygen.
Oxidase reagent contains a chromogenic reducing agent which is a compound that
changes its color when it becomes oxidized.
If the bacteria produced Cytochrome C Oxidase, the oxidase reagent will turn blue
or dark purple color within 10 to 15 seconds.
This test almost perform for to identify oxidase negative Enterobacteriaceae and
oxidase positive Pseudomadaceae .
Objective: To identify Cytochrome C Oxidase bacteria
Requirements:
1. Fresh 24 hours Bacterial Sample
2. Distilled water
3. Oxidase reagent ( Tetra methyl-P-Phenylenediamine Dihydrochloride )
4. Test tubes
5. Test tubes stand
6. Filter paper
7. Wire loop
8. Glass slide
9. Distilled water
10. Glass slides
11. Sprite lamp / Burner
12. Slides stray
13. Dropper / Micro pipette
14. LFH
15. Gloves
16. Mask

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17. Ethanol
Procedure:
1. Take test 2 ml distilled water in the test tube.
18. Put a minute amount of Oxidase reagent ( Tetra methyl-P-
Phenylenediamine Dihydrochloride ), make its solution , shakes the test
tubes until changed its color into light color.
2. Replaced filter paper on the slide and dropped a drop of solution from the
test tube on the filter paper.
3. By using wire loop pick a fresh colony from the growth cultured and mixed
with drop on the filter paper.
4. Within 10 to 15 seconds bacteria will reacts with the drop solution on the
filter paper and will changed its color into blue purple, and the result will be
considered as Oxidase Positive.
(4) INDOLE TEST:
Indole test is a biochemical test which is perform for identification of gram
Negative bacteria.
Bacteria which express the enzyme called Tryptophanase which hydrolyze amino
acid ( Tryptophane ) into indole , pyruvic acid and ammonia.
Presence of indole can be shown by means of Kovac’s reagent in the so called
Indole test. Kovac’s reagent contains P-dimethyl amino benzal dehyde, which
forms a red complex with indole.
Objective: To identify Tryptophanase enzyme producing bacteria.
Requirements:
1. Broth Media ( Peptone Water )
2. Fresh 24 hours Bacterial Sample
3. Distilled water
4. Kovac’s reagent
5. Test Tubes
6. Test tubes stand

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7. Digital balance
8. Flask
9. Graduate Cylinder
10. Autoclave
11. Incubator
12. Heat plate
13. Aluminum foil
14. Cotton
15. Wire loop
16. Dropper / Micro pipette
17. Sprite lamp / Burner
18. Match bar
19. Gloves
20. Mask
21. Ethanol
22. LFH
Procedure:
1. Prepare peptone water media in the flask with respect to the numbers of test
tubes.
2. After preparation the media , distributed the media in the test tubes , covered
the test tubes with cotton and aluminum foil and allow for autoclaving.
3. After autoclaving allow the test tubes to cool .
4. Next, by using wire loop pick a bacterial inoculum from the broth cultured
and deep in the test tubes and labeled the test tubes.
5. After inoculation , incubated the test tubes at 37˚C for 24 hours.
6. After 24 hours absorb the test tubes ,the bacterial growth will appear inside
the test tubes.
7. Next apply ( .5 ml) Kovac’s reagent in the growth test tubes . within 10
minutes a red ring will formed at the top of growth test tube .
8. Result:
 If the ring is formed its mean the result is positive , because the
concern species produced an enzyme call Tryptophanase.
 If there is no ring formation or formed green ring formation its mean
the result is negative , because the concern species do not have the

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ability to produced Tryptophanase enzyme to break the a Tryptophan amino acid in
to ammonia and pyruvic acid…


(5) CITRATE TEST:
Objective: To determines the ability of microorganism to utilize Citrate
Citrate Test is a biochemical test which is use for Gram Negative bacteria. Gram
negative bacteria used citrate as sole carbon source, and changed the color of the
media from green to deep blue.
This test is often used to differentiate in between the numbers of Enterobacteriae.
In bacteria having capability of utilizing citrate as a carbon source, the enzymes
Citrase hydrolyzes citrate in to Oxaoloacetic acid and acetic acid.
Oxaoloacetic acid is then hydrolyzed into Pyruvic acid and CO2 . The products of
the reaction are acetate, Lactic acid, formic acid etc.
If CO2 is produced, it reacts with the components of the medium to produce an
Alkaline compound I.e Sodium Carbonate (Na2CO3 ).
The Alkaline pH turns the pH indicator ( Bromthymol blue ) from green to blue.
This is a positive result ( the tube on the right is citrate positive) .

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Klebsiella Pneumoniae and proteus mirabilis are the examples of Citrate positive
organisms.
Requirements:
1. Citrate Media
2. Fresh 24 hours Bacterial Sample
3. Distilled water
4. Test tubes
5. Test tubes stand
6. Digital balance
7. Heat plate
8. Flask
9. Beaker
10. Graduate Cylinder
11. Autoclave
12. Incubator
13. Aluminum foil
14. Cotton
15. Wire loop
16. Sprite lamp / Burner
17. Match bar
18. Gloves
19. Mask
20. Permanent marker
21. LFH
22. Ethanol
23. Soap
Procedure:
1. Prepare Citrate Media in the flask and allow it on the heat plate to dissolved
it completely.
2. Pouring the media in the test tubes, covered the test tubes with cotton and
aluminum foil and allow for autoclaving.

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3. After autoclaving keep the test tubes at 30 Angle and make its slant and
allow it to solidify.
4. After slant formation with the help of straight wire loop pick up a fresh
bacterial colony and Inoculate by first stabbing through the center of the
medium to the bottom of the test tube and then streaking on the surface of
the agar slant and labeled the test tubes.
5. After inoculation replace the test tubes in the incubator at 37˚C for 24 hours.
6. After 24 hours absorb the test tubes.
7. Result:
 Positive: The color of the citrate medium will changed from green to
blue as well as production of cracks in the butt deep due to CO2
production.
 Negative: There will be no changing in test tubes medium.

Triple Sugar Iron Agar (TSI) test
Principle:
This medium contains three sugars namely:
a. 10 parts lactose
b. 10 parts sucrose
c. 1 part glucose and peptone

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and also iron; and it contains Agar as solidifying agent (TSI is a semi solid media
having slant and butt).
TSI agar is used to determine whether a gram negative rod shape bacteria utilizes
glucose and lactose or sucrose fermentatively and forms hydrogen sulphide (H2S).
Phenol red and ferrous sulphate serves as indicators of acidification and H2S
formation, respectively. The formation of CO2 and H2 is indicated by the presence
of bubbles or cracks in the agar or by separation of the agar from the sides or
bottom of the tube. The production of H2S requires an acidic environment and is
indicated by blackening of the butt of the medium in the tube.
Objective:
TSI is used for determination of carbohydrate fermentation and H ydrogen sulphide
(H
2S) production and the identification of gram negative Bacilli
Requirements:
1. TSI Media
2. Fresh 24 hours Bacterial Sample
3. Distilled water
4. Test tubes
5. Test tubes stand
6. Digital balance
7. Flask
8. Beaker
9. Graduate Cylinder
10. Autoclave
11. Incubator
12. Aluminum foil
13. Cotton
14. Wire loop
15. Sprite lamp / Burner
16. Match bar
17. Gloves
18. Mask
19. Permanent marker

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20. LFH
21. Ethanol
22. Soap
Procedure:
1. Prepare TSI media in the flask, purring the media in the test tubes, covered
the test tubes by cotton and aluminum foil and autoclave it at 121˚C for 45
minutes at 15 psi.
2. After autoclaving bring the test tubes in the LFH , keep it at 30 Angle and
make its slant.
3. After solidification with the help of straight wire loop pick up a fresh
bacterial colony and Inoculate by first stabbing through the center of the
medium to the bottom of the test tube and then streaking on the surface of
the agar slant and labeled the test tubes.
4. After inoculation incubated the test tubes at 37 ˚C for 24 hours.
5. After incubation absorb the test tubes and noted the result.
Result interpretation:
A. Alkaline slant/no change in the butt (K/NC) = Glucose, lactose and sucrose
non‐utilizer(alkaline slant/alkaline butt) [figure: 1(d)]
B. Alkaline slant/acid butt (K/A) = Glucose fermentation only. [figure: 1(b)]
C. Acid slant/acid butt (A/A), with gas production = Glucose, sucrose, and/or
lactose fermenter. [figure: 1(a)]
Alkaline slant/acid butt (K/A), H2S production = Glucose fermentation only.
[figure: 1(c)] A B C D

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2
nd
Fagure

Quality control:
 A/A, with gas: E. coli
 K/A, H2S: Salmonella typhi
 K/NC: Pseudomonas aeruginosa
Some examples of TSI Agar Reactions:
Name of the
organisms 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 (-)
(6) UREASE TEST:
Urease is a biochemical test which is used to identify bacteria having capability of
hydrolyzing urea by using urease enzyme. Its commonly used to distinguish the

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genus proteus from other enteric bacteria. The hydrolysis of urea forms the weak
base, ammonia, as one of its products . this weak base raises the pH of the media
above 8.4 and the pH indicator, phenol red, turns from yellow to pink. Proteus
mirabilis is a rapid hydrolyzer of urea.
Objective: To determined the urease producing bacteria.
Requirements:
1. Urea Agar
2. Bacterial Sample
3. Distilled water
4. Test tubes
5. Test tubes stand
6. Digital balance
7. Flask
8. Beaker
9. Graduate Cylinder
10. Autoclave
11. Incubator
12. Aluminum foil
13. Cotton
14. Wire loop
15. Sprite lamp / Burner
16. Match bar
17. Gloves
18. Mask
19. Permanent marker
20. LFH
21. Ethanol
22. Soap
Procedure:
1. Prepare Area Agar media in the flask , purring in the test tubes, covered the
test tubes with cotton and aluminum foil.

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2. After pouring allow the test tubes for autoclaving at 121˚C for 45 minutes at
15 psi.
3. After autoclaving replaced the test tubes in the LFH, keep at 30 Angle and
make its slant.
4. After solidification with the help of straight wire loop pick up a inoculum
from fresh bacterial cultured and Inoculate by first stabbing through the
center of the medium to the bottom of the test tube and then streaking on
the surface of the agar slant and labeled the test tubes.
5. After inoculation allow the test tubes in the incubator , and incubated the test
tubes at 37 ˚C for 18 to 24 hours.
6. After 24 hours absorb the test tubes.
7. Result:
 If the medium of the test tube is changed from yellow into pink , the
result should be positive.
 If there is no change in the test tube medium, the result should me
consider as negative.


Don't compare yourself with anyone in this world…if you do so, you are insulting yourself.
Thousands of candles can be lit from a single candle and the life of the candle will not be shortened
Happiness never decreases from being shared.
"If you want to be successful, find someone who has achieved the results you want and copy what they do
and you'll achieve the same results."

Demonstrated By:
 Sir Ghadir Ali
 Madam Azra
 Madam Saira

Prepared By Amjad Khan Afridi