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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 StainingB. 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 NegativePeptidoglycan 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

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Porins proteins Absent Present

Permeability of molecules More permeable Less permeable

MATERIALS :1. 24 hours fresh bacterial cultural2. Crystal violet3. Iodine4. De-colorizer ( Ethyl alcohol)5. Safranin6. Oil ( Immersion Oil )7. Distilled water8. Wire loop9. Glass slides10.Slides covers11.Sprite lamp / Burner12.Slides stray13.Bibulous paper / absorbent paper14.Beaker15.Gloves16.Mask17.Permanent marker18.Ethanol19.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.

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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.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.

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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 :

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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(B) BIOCHEMICAL TESTS

For the identification of gram positive and gram negative bacteria we use the following biochemical tests.

1. Catalase Test2. Coagulase Test3. Oxidase Test4. Indole Test5. Citrate Test6. TSI ( Triple Sugar Iron) Test7. Urease Test

(1) CATALASE TEST

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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 Tubes6. Test tube stand7. Bunsen burner8. Syringe 9. Glass slides

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.

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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 testb. The Tube coagulase test

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

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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. 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 test4. bacteria is added.5. To the tube labeled positive control, 0.2 ml of overnight broth culture of known

S.aureus is added6. 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.

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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

(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.

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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 water3. Oxidase reagent ( Tetra methyl-P-Phenylenediamine Dihydrochloride )4. Test tubes5. Test tubes stand6. Filter paper7. Wire loop8. Glass slide9. Distilled water10.Glass slides11.Sprite lamp / Burner12.Slides stray13.Dropper / Micro pipette 14.LFH15.Gloves16.Mask17.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.

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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 water4. Kovac’s reagent 5. Test Tubes6. Test tubes stand7. Digital balance8. Flask9. Graduate Cylinder10. Autoclave11. Incubator12. Heat plate13. Aluminum foil 14. Cotton15. Wire loop16. Dropper / Micro pipette 17. Sprite lamp / Burner18. Match bar

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19. Gloves20. Mask21. Ethanol22. 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

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

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(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) .

Klebsiella Pneumoniae and proteus mirabilis are the examples of Citrate positive organisms.

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1. Citrate Media 2. Fresh 24 hours Bacterial Sample 3. Distilled water 4. Test tubes5. Test tubes stand6. Digital balance7. Heat plate8. Flask9. Beaker10. Graduate Cylinder11. Autoclave12. Incubator13. Aluminum foil 14. Cotton15. Wire loop16. Sprite lamp / Burner17. Match bar18. Gloves19. Mask20. Permanent marker21. LFH22.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.

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

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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 lactoseb. 10 parts sucrosec. 1 part glucose and peptone

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).

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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 Hydrogen sulphide (H2S) production and the identification of gram negative Bacilli

Requirements:

1. TSI Media 2. Fresh 24 hours Bacterial Sample 3. Distilled water 4. Test tubes5. Test tubes stand6. Digital balance7. Flask8. Beaker9. Graduate Cylinder10. Autoclave11. Incubator12. Aluminum foil 13. Cotton14. Wire loop15. Sprite lamp / Burner16. Match bar17. Gloves18. Mask19. Permanent marker20. LFH21.Ethanol 22. Soap

Procedure:

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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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2nd 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 genus proteus from other enteric bacteria. The hydrolysis of urea forms the weak

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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 Agar2. Bacterial Sample 3. Distilled water 4. Test tubes5. Test tubes stand6. Digital balance7. Flask8. Beaker9. Graduate Cylinder10. Autoclave11. Incubator12. Aluminum foil 13. Cotton14. Wire loop15. Sprite lamp / Burner16. Match bar17. Gloves18. Mask19. Permanent marker20. LFH21.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.

2. After pouring allow the test tubes for autoclaving at 121˚C for 45 minutes at 15 psi.

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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

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