MICROBIOLOGY LAB TESTS. The following microbiology tests will be performed in lab this semester Gram stain Motility EMB ( Eosin Methylene blue agar)

MICROBIOLOGY LAB MICROBIOLOGY LAB TESTSTESTS

The following microbiology tests will be The following microbiology tests will be performed in lab this semesterperformed in lab this semester

Gram stainGram stain MotilityMotility EMB ( Eosin Methylene blue agar)EMB ( Eosin Methylene blue agar) MacConkey agarMacConkey agar Carbohydrate fermentation ( glucose, lactose, Carbohydrate fermentation ( glucose, lactose,

sucrose)sucrose) Gelatine liquefaction testGelatine liquefaction test Mannitol saltMannitol salt IndolIndol SIM ( indol, motility,sulfur reduction) SIM ( indol, motility,sulfur reduction)

IMViCIMViC tests tests ((IndoleIndole , ,Methyl Methyl RedRed,Voges-,Voges-

Proskauer (VP) test,Proskauer (VP) test,Citrate TestCitrate Test)) Triple sugar Iron AgarTriple sugar Iron Agar Skim Milk agarSkim Milk agar CatalaseCatalase

Gram stainGram stain

Purpose:Purpose: The Gram stain is the most The Gram stain is the most commonly used differential stain for commonly used differential stain for determining cell morphology.determining cell morphology.

The gram stain divides most bacteria into The gram stain divides most bacteria into two main groups based on the amount of two main groups based on the amount of the sugar: the sugar: peptidoglycanpeptidoglycan present in the present in the bacteria cell wall.bacteria cell wall.

Gram stainGram stainProcedureProcedure

Procedure:Procedure:1.1. 1. clean your slide1. clean your slide2.2. Put ½ drop of water on the slidePut ½ drop of water on the slide3.3. Using a bacteria loop add bacteria to the slideUsing a bacteria loop add bacteria to the slide4.4. Let slide air dryLet slide air dry5.5. Heat fix the slideHeat fix the slide6.6. Add crystal violet to slide and let sit for 2 Add crystal violet to slide and let sit for 2

minutes.minutes.7.7. Wash off under waterWash off under water8.8. Add iodine to slide and let sit for 2 minutesAdd iodine to slide and let sit for 2 minutes

Procedure continue:Procedure continue:

9.9. Wash off under waterWash off under water10.10. THIS IS THE MOST IMPORTANT STEP!! THIS IS THE MOST IMPORTANT STEP!!

Holding slide at an angle, carefully add Holding slide at an angle, carefully add ethyl alcohol for 8 seconds ethyl alcohol for 8 seconds

11.11. After 8 seconds are up, wash off alcohol After 8 seconds are up, wash off alcohol under water.under water.

12.12. Add safranin red for at least 2 minutesAdd safranin red for at least 2 minutes13.13. Wash off and blot dryWash off and blot dry

14.14. Examine under microscopeExamine under microscope

Gram Positive bacteriaGram Positive bacteria

Gram positiveGram positive bacteria bacteria stain purplestain purple and and have a thick layer of peptidoglycan in the have a thick layer of peptidoglycan in the cell wallcell wall

Gram Negative bacteriaGram Negative bacteria Gram negativeGram negative bacteria bacteria stain pinkstain pink and and

have a thin layer of peptidoglycan in the have a thin layer of peptidoglycan in the cell wallcell wall

MotilityMotility

PurposePurpose: “The : “The movement of bacteria is movement of bacteria is detected by use of a detected by use of a medium with low agar medium with low agar concentration. concentration.

The medium is The medium is inoculated with an inoculated with an inoculating needleinoculating needle

Reading the test: Reading the test: An An organism that is motile organism that is motile will grow out into the will grow out into the surrounding medium surrounding medium away from the stab line.away from the stab line.

MotilityMotility Test Tube ResultsTest Tube Results

Test tubeTest tube 1: Motile 1: Motile

Test TubeTest Tube 2:Non- 2:Non-motile motile

Test TubeTest Tube 3 3：： Motile Motile

Motility TestMotility Test

EMB ( Eosin Methylene EMB ( Eosin Methylene blue agar)blue agar)

PurposePurpose: This agar a is selective and : This agar a is selective and differential medium used for isolation differential medium used for isolation

and differentiation among and differentiation among Enterobacteriaceae.Enterobacteriaceae.

EMB ( Eosin Methylene EMB ( Eosin Methylene blue agar)blue agar)

EMB is selective for gram negative EMB is selective for gram negative bacteria meaning bacteria meaning only gram only gram

negative bacteria will grownegative bacteria will grow on this on this medium.medium.

EMB ( Eosin Methylene blue agar)EMB ( Eosin Methylene blue agar)

EMB also contains lactose.EMB also contains lactose. Organisms which Organisms which do not ferment lactose do not ferment lactose

will have colorless colonieswill have colorless colonies Organisms which Organisms which do ferment lactose will do ferment lactose will

have pink colonieshave pink colonies.. E. coli develops a metallic greenE. coli develops a metallic green sheet sheet

of coloniesof colonies

EMB with E. coliEMB with E. coli

Organisms which Organisms which do ferment do ferment lactose will have pink colonieslactose will have pink colonies..

.

Organisms which Organisms which do ferment do ferment lactose will have pink colonieslactose will have pink colonies

MacConkey agarMacConkey agar

PurposePurpose: MacConkey is both : MacConkey is both selective and differential used to selective and differential used to

differentiate between gram negative differentiate between gram negative and gram positive bacteria. and gram positive bacteria.


MacConkey is selective for gram MacConkey is selective for gram negative bacteria meaning negative bacteria meaning only only

gram negative bacteria will growgram negative bacteria will grow on on this medium.this medium.


MacConkey also contains lactose.MacConkey also contains lactose. Organisms which Organisms which do not ferment lactose do not ferment lactose

will have colorless colonieswill have colorless colonies Organisms which Organisms which do ferment lactose will do ferment lactose will

have pink colonieshave pink colonies

Organisms which Organisms which do not ferment do not ferment lactose will have colorless colonieslactose will have colorless colonies

Organisms which Organisms which do ferment do ferment lactose will have pink colonieslactose will have pink colonies

MacConkey Agar

Left: Shigella

- result

Right: Escherichia

+ result


1. Which plate could have 1. Which plate could have E.E. colicoli on on it?it?

2. Which plate shows the presence 2. Which plate shows the presence of a lactose-fermenter?of a lactose-fermenter?

3. Which plate could have 3. Which plate could have SalmonellaSalmonella on it? on it?

4. Which plate shows the presence 4. Which plate shows the presence of aof a

lactose non-fermenter?lactose non-fermenter?

Answers:

1.A3. B

2. A4. B

A

B

Carbohydrate fermentationCarbohydrate fermentation ( ( glucose, lactose, sucrose)glucose, lactose, sucrose)

Purpose:Purpose: This test is used to determine the This test is used to determine the ability of an organism to ferment various simple ability of an organism to ferment various simple carbohydrates ( sugars)carbohydrates ( sugars)

A small tube ( A small tube ( Durham tubeDurham tube) is inverted and ) is inverted and place in each larger tube of liquid medium place in each larger tube of liquid medium

The inverted tube is able to trap any gas The inverted tube is able to trap any gas produced. produced.

The indicator, phenol red will turn yellow below a The indicator, phenol red will turn yellow below a pH of 6.8 9 showing fermentation) and darker pH of 6.8 9 showing fermentation) and darker pinkish-red above pH 7.4 ( showing no pinkish-red above pH 7.4 ( showing no fermentation)fermentation)


Reading the Test: Reading the Test:

Acid: (yellowAcid: (yellow): ): Positive Positive for metabolizing for metabolizing sugar + sugar + ( ( a color change has happeneda color change has happened))

Acid, GasAcid, Gas: ( yellow plus gas bubble): : ( yellow plus gas bubble): Positive for metabolizing sugar and gas Positive for metabolizing sugar and gas production (+) production (+) ( ( color change plus bubblescolor change plus bubbles) )

Negative: NO color changeNegative: NO color change


Carbohydrate Fermentation TestsGlucose w/ Durham Fermentation Tube

Carbohydrate Fermentation TestsSucrose Fermentation

Gelatine liquefaction testGelatine liquefaction test

Purpose:Purpose: This test is used to determine the This test is used to determine the ability of the bacteria to produce ability of the bacteria to produce gelatinasesgelatinases that that digest and liquefy gelatin.digest and liquefy gelatin.

Reading Test:Reading Test: Once the tubes are inoculated with the bacteria Once the tubes are inoculated with the bacteria

and incubated, they will cause the gelatin to melt. and incubated, they will cause the gelatin to melt. In order to determine whether or not the reaction In order to determine whether or not the reaction

has taken place, the incubated tubes must be has taken place, the incubated tubes must be placed on ice until the control tube is once again placed on ice until the control tube is once again solid.solid.

Gelatine liquefaction testGelatine liquefaction test

Positive TestPositive Test Negative TestNegative Test

Mannitol saltMannitol salt

Purpose: Mannitol salt agar is both Purpose: Mannitol salt agar is both selective and differential. selective and differential.

Principle: Mannitol salt helps determine Principle: Mannitol salt helps determine two characteristics of bacteria, whether two characteristics of bacteria, whether they are salt tolerant or not, and whether they are salt tolerant or not, and whether they are able to ferment mannitolthey are able to ferment mannitol

Mannitol salt used to id Mannitol salt used to id Staphlococcus bacteriaStaphlococcus bacteria

Mannitol Mannitol saltsalt agar is a selective and differential agar is a selective and differential primary culture medium useful in recovery and primary culture medium useful in recovery and identification of staphylococci from specimens identification of staphylococci from specimens containing mixed flora. containing mixed flora.

The high salt concentration (7.5%) inhibits most The high salt concentration (7.5%) inhibits most gram-negative and gram-positive bacteria gram-negative and gram-positive bacteria except except Staphylococcus speciesStaphylococcus species

The The Staphylococcus aureusStaphylococcus aureus ferments mannitol and ferments mannitol and turns the medium yellow. The turns the medium yellow. The Serratia marcescensSerratia marcescens

does not grow because of the high salt contentdoes not grow because of the high salt content

Streptococcus agalactiaeStreptococcus agalactiae does not grow on MSA does not grow on MSA because of the high salt content. because of the high salt content. Staphylococcus Staphylococcus epidermidisepidermidis grows but does not ferment mannitol. grows but does not ferment mannitol.

IndolIndol

Purpose: The indole test is used to identify Purpose: The indole test is used to identify bacteria capable of producing indole.bacteria capable of producing indole.

Reading the TestReading the Test: : When When Kovac’s solution is addedKovac’s solution is added to the to the

inoculated tube, a inoculated tube, a red layerred layer will form when will form when indol is present indol is present = positive test.= positive test.

No red layer = negative test No red layer = negative test

IndolIndolWith the Kovac’s reagent addedWith the Kovac’s reagent added::

Positive TestPositive Test Negative TestNegative Test

SIM TestSIM Test(Indol, motility, sulfur reduction) (Indol, motility, sulfur reduction)

Purpose: The SIM test performs three Purpose: The SIM test performs three different tests: motility, indole and sulfur different tests: motility, indole and sulfur reduction.reduction.

Motility and indole have been mentioned Motility and indole have been mentioned earlierearlier

Sulfur reductionSulfur reduction: This test is used to : This test is used to identify those bacteria capable of reducing identify those bacteria capable of reducing sulfur. sulfur.

SIM TestSIM Test(, sulfur reduction portion)(, sulfur reduction portion)

Black precipitate indicates a Black precipitate indicates a positive test (for H2S positive test (for H2S production)production)

No precipitate means No precipitate means a negative testa negative test

IMViCIMViC tests tests

The The IMViCIMViC tests are a group of individual tests are a group of individual tests used in microbiology lab testing to tests used in microbiology lab testing to identify an organism in the coliform group.identify an organism in the coliform group.

A coliform is a gram negative, aerobic or A coliform is a gram negative, aerobic or facultative aerobic rod which produces gas facultative aerobic rod which produces gas from lactose within 48 hours. from lactose within 48 hours.

The presence of some coliforms indicate The presence of some coliforms indicate fecal contamination.fecal contamination.

These four tests includeThese four tests include

IndoleIndole test test ( discussed earlier) ( discussed earlier) Methyl RedMethyl Red test test Voges-Proskauer (VP) test * Voges-Proskauer (VP) test * Citrate TestCitrate Test

Indole TestIndole Test

broth with tryptophan

IMViC Series

Methyl RedMethyl Red test and Voges- test and Voges-Proskauer (VP) test * Proskauer (VP) test *

These tests both use the same broth for These tests both use the same broth for bacterial growth. bacterial growth.

The broth is called The broth is called MRVP broth.MRVP broth. After growth, the broth is separated into After growth, the broth is separated into

two different tubes, one for the Methyl Red two different tubes, one for the Methyl Red (MR) test and one for the Voges-(MR) test and one for the Voges-Proskauer (VP) test. Proskauer (VP) test.

Methyl RedMethyl Red

The pH indicator The pH indicator Methyl Red is added Methyl Red is added to one tube and a red to one tube and a red color appears at pH's color appears at pH's lower than 4.2, and lower than 4.2, and indicated positive test. indicated positive test.

Methyl Red TestMethyl Red TestIMViC Series

VP testVP test

The reagents used for the VP test are Barritt's A The reagents used for the VP test are Barritt's A (alpha-napthol) and Barritt's B (potassium (alpha-napthol) and Barritt's B (potassium hydroxide). hydroxide).

When these reagents are added to a broth in When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn which acetyl methyl carbinol is present, they turn a pink-burgundy color (a positive VP test). a pink-burgundy color (a positive VP test).

This color may take 20 to 30 minutes to develop. This color may take 20 to 30 minutes to develop. E. coli E. coli does not produce acetyl methyl carbinol, does not produce acetyl methyl carbinol,

Voges-Proskauer TestVoges-Proskauer Test

Leave uncapped for 15-20 minutes……..

IMViC Series

MRVP broth

VP testVP test

..

Citrate TestCitrate Test

This test uses Simmon's citrate agar to This test uses Simmon's citrate agar to determine the ability of a determine the ability of a

microorganism to use citrate as its sole microorganism to use citrate as its sole carbon source. carbon source.

Simmons Citrate TestSimmons Citrate Test

The citrate agar is green before The citrate agar is green before inoculation, and turns blue as a positive inoculation, and turns blue as a positive test indicatortest indicator

Citrate Utilization TestCitrate Utilization Test

- +

IMViC Series

IMViC testsIMViC tests

These IMViC tests are useful for differentiating These IMViC tests are useful for differentiating the family the family EnterobacteriaceaeEnterobacteriaceae, especially when , especially when used alongside the used alongside the Urease testUrease test..

Except for the lowercase “i”, which is added for Except for the lowercase “i”, which is added for ease of pronunciation, each of the letters in ease of pronunciation, each of the letters in “IMViC” stands for one of these tests. “I” is for “IMViC” stands for one of these tests. “I” is for indole; “M” is for methyl red; “V” is for Voges-indole; “M” is for methyl red; “V” is for Voges-Proskauer, and “C” is for citrate.Proskauer, and “C” is for citrate.

Triple sugar Iron AgarTriple sugar Iron Agar

The tube is inoculated by stabbing into the The tube is inoculated by stabbing into the agar butt (bottom of the tube) with an agar butt (bottom of the tube) with an inoculating wire and then streaking the inoculating wire and then streaking the slant in a wavy pattern.slant in a wavy pattern.

Results are read at 18 to 24 hours of Results are read at 18 to 24 hours of incubation. incubation.


When there is a yellow butt and a red slant When there is a yellow butt and a red slant that indicates that the organism can that indicates that the organism can ferment glucose.ferment glucose.

The change in color from red to yellow is The change in color from red to yellow is due to the change in pH as lactic acid is due to the change in pH as lactic acid is produced.produced.

The pH indicator used is Phenol Red The pH indicator used is Phenol Red


Nonfermentative Gram-negative Bacilli

Positive testPositive test

Triple Sugar Iron

-/+/+

Starch AgarStarch Agar Starch Agar is basically an all-purpose medium Starch Agar is basically an all-purpose medium A plate of this medium is single streaked or spot A plate of this medium is single streaked or spot

inoculated with the organism to be tested. After inoculated with the organism to be tested. After incubation (preferably for several days) the incubation (preferably for several days) the medium is flooded with Gram's iodine medium is flooded with Gram's iodine

The iodine reacts with starch to form a dark The iodine reacts with starch to form a dark blue-colored complex.blue-colored complex.

Any Any clear area around the growthclear area around the growth of the of the culture after the addition of the iodine indicates culture after the addition of the iodine indicates the the breakdown of starchbreakdown of starch by the organism due by the organism due to its production of to its production of amylaseamylase, an extracellular , an extracellular enzyme. enzyme.

Starch Agar and the Amylase Test Starch Agar and the Amylase Test Starch Agar and the Amylase Test Starch Agar and the Amylase Test

The clear (non-blue) area The clear (non-blue) area around the culture on the around the culture on the left indicates the absence left indicates the absence of intact starch – and of intact starch – and therefore a positive therefore a positive reaction for the amylase reaction for the amylase test. test.

The blue color extends up The blue color extends up to the culture on the right to the culture on the right which is thus seen to be which is thus seen to be unable to break down unable to break down starch – a negative starch – a negative reaction for this test. reaction for this test.

Skim Milk agarSkim Milk agar

Skim Milk agar is used to detect the Skim Milk agar is used to detect the presence of caseinase.presence of caseinase.

Caseinase is an enzyme that catalyzes Caseinase is an enzyme that catalyzes the breakdown of casein into its individual the breakdown of casein into its individual amino acids.amino acids.


Negative testNegative test


This bacteria This bacteria produces produces caseinase which caseinase which has resulted in has resulted in the breakdown the breakdown of the casein on of the casein on either side of the either side of the bacterial growth. bacterial growth. This is seen as a This is seen as a clear zoneclear zone

CatalaseCatalase

Catalase Catalase is an enzyme that converts is an enzyme that converts hydrogen peroxide into water and oxygen. hydrogen peroxide into water and oxygen.

The presence of catalase can be easily The presence of catalase can be easily detected by the slide method.detected by the slide method.

A drop of 3 percent hydrogen peroxide is put A drop of 3 percent hydrogen peroxide is put on a slide and the bacteria is emulsified in on a slide and the bacteria is emulsified in the drop. the drop.

The presence of bubbles is evidence of the The presence of bubbles is evidence of the production of oxygenproduction of oxygen

CatalaseCatalase

What is the name of the lab procedure demonstrated here?

Answer: Stabbing the butt!

I M V

C

U

Use the chart and/or the flow chart your lab manual to figure out who I am!

Answer: Escherichia coli

I M V C

U

Use the and/or the flow chart on of your lab manual to figure out who I am!

Answer: Serratia marcesens

Use the chart and/or the flow chart of your lab manual to figure out who I am!

I M V C

Answer: Enterobacter aerogenes

U

Documents

MICROBIOLOGY LAB TESTS. The following microbiology tests will be performed in lab this semester Gram stain Motility EMB ( Eosin Methylene blue agar)