MICROBIOLOGY LAB TESTS

The following microbiology tests will be performed in lab this semester Gram stain Motility EMB ( Eosin Methylene blue agar) MacConkey agar Carbohydrate fermentation ( glucose, lactose, sucrose) Gelatine liquefaction test Mannitol salt Indol SIM ( indol, motility,sulfur reduction)

IMViC tests (Indole ,Methyl Red,Voges-Proskauer (VP) test,Citrate Test) Triple sugar Iron Agar Skim Milk agar Catalase

Gram stain Purpose: The Gram stain is the most commonly used differential stain for determining cell morphology. The gram stain divides most bacteria into two main groups based on the amount of the sugar: peptidoglycan present in the bacteria cell wall.

Gram stain Procedure Procedure: 1. clean your slide Put ½ drop of water on the slide Using a bacteria loop add bacteria to the slide Let slide air dry Heat fix the slide Add crystal violet to slide and let sit for 2 minutes. Wash off under water Add iodine to slide and let sit for 2 minutes

Procedure continue: Wash off under water THIS IS THE MOST IMPORTANT STEP!! Holding slide at an angle, carefully add ethyl alcohol for 8 seconds After 8 seconds are up, wash off alcohol under water. Add safranin red for at least 2 minutes Wash off and blot dry Examine under microscope

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

Gram Negative bacteria Gram negative bacteria stain pink and have a thin layer of peptidoglycan in the cell wall

Motility Purpose: “The movement of bacteria is detected by use of a medium with low agar concentration. The medium is inoculated with an inoculating needle Reading the test: An organism that is motile will grow out into the surrounding medium away from the stab line.

Motility Test Tube Results Test tube 1: Motile Test Tube 2:Non-motile

Motility Test 11

EMB ( Eosin Methylene blue agar) Purpose: This agar a is selective and differential medium used for isolation and differentiation among Enterobacteriaceae.

EMB ( Eosin Methylene blue agar) EMB is selective for gram negative bacteria meaning only gram negative bacteria will grow on this medium.

EMB ( Eosin Methylene blue agar) EMB also contains lactose. Organisms which do not ferment lactose will have colorless colonies Organisms which do ferment lactose will have pink colonies. E. coli develops a metallic green sheet of colonies

EMB with E. coli

Organisms which do ferment lactose will have pink colonies.                                                                                                                                                                                                                                                .                                                                                                                                                            

Organisms which do ferment lactose will have pink colonies

MacConkey agar Purpose: MacConkey is both selective and differential used to differentiate between gram negative and gram positive bacteria.

MacConkey agar MacConkey is selective for gram negative bacteria meaning only gram negative bacteria will grow on this medium.

MacConkey agar MacConkey also contains lactose. Organisms which do not ferment lactose will have colorless colonies Organisms which do ferment lactose will have pink colonies

Organisms which do not ferment lactose will have colorless colonies

Organisms which do ferment lactose will have pink colonies

MacConkey Agar Left: Shigella - result Right: Escherichia + result 23

A B 2. A 4. B A 3. B Answers: MacConkey agar 1. Which plate could have E. coli on it? 2. Which plate shows the presence of a lactose-fermenter? 3. Which plate could have Salmonella on it? 4. Which plate shows the presence of a lactose non-fermenter?                                                                                             A B 2. A 4. B A 3. B Answers: 24

Carbohydrate fermentation ( glucose, lactose, sucrose) Purpose: This test is used to determine the ability of an organism to ferment various simple carbohydrates ( sugars) A small tube ( Durham tube) is inverted and place in each larger tube of liquid medium The inverted tube is able to trap any gas produced. The indicator, phenol red will turn yellow below a pH of 6.8 9 showing fermentation) and darker pinkish-red above pH 7.4 ( showing no fermentation)

Carbohydrate fermentation ( glucose, lactose, sucrose) Reading the Test: Acid: (yellow): Positive for metabolizing sugar + ( a color change has happened) Acid, Gas: ( yellow plus gas bubble): Positive for metabolizing sugar and gas production (+) ( color change plus bubbles) Negative: NO color change

Carbohydrate fermentation ( glucose, lactose, sucrose)

Carbohydrate Fermentation Tests Glucose w/ Durham Fermentation Tube 28

Carbohydrate Fermentation Tests Sucrose Fermentation 29

Gelatine liquefaction test Purpose: This test is used to determine the ability of the bacteria to produce gelatinases that digest and liquefy gelatin. Reading Test: Once the tubes are inoculated with the bacteria and incubated, they will cause the gelatin to melt. In order to determine whether or not the reaction has taken place, the incubated tubes must be placed on ice until the control tube is once again solid.

Gelatine liquefaction test                                       Gelatine liquefaction test                                       Positive Test Negative Test                                                                                                                                                                                                  [                                      

Mannitol salt Purpose: Mannitol salt agar is both selective and differential. Principle: Mannitol salt helps determine two characteristics of bacteria, whether they are salt tolerant or not, and whether they are able to ferment mannitol

Mannitol salt used to id Staphlococcus bacteria Mannitol salt agar is a selective and differential primary culture medium useful in recovery and identification of staphylococci from specimens containing mixed flora. The high salt concentration (7.5%) inhibits most gram-negative and gram-positive bacteria except Staphylococcus species

The Staphylococcus aureus ferments mannitol and turns the medium yellow.  The Serratia marcescens does not grow because of the high salt content

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

Indol Purpose: The indole test is used to identify bacteria capable of producing indole. Reading the Test: When Kovac’s solution is added to the inoculated tube, a red layer will form when indol is present = positive test. No red layer = negative test

Indol With the Kovac’s reagent added: Positive Test Negative Test

SIM Test (Indol, motility, sulfur reduction) Purpose: The SIM test performs three different tests: motility, indole and sulfur reduction. Motility and indole have been mentioned earlier Sulfur reduction: This test is used to identify those bacteria capable of reducing sulfur.

SIM Test (, sulfur reduction portion)                                                                      SIM Test (, sulfur reduction portion)                        Black precipitate indicates a positive test (for H2S production) No precipitate means a negative test                               

IMViC tests The IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. A coliform is a gram negative, aerobic or facultative aerobic rod which produces gas from lactose within 48 hours. The presence of some coliforms indicate fecal contamination.

These four tests include Indole test ( discussed earlier) Methyl Red test Voges-Proskauer (VP) test * Citrate Test

IMViC Series Indole Test broth with tryptophan 42

Methyl Red test and Voges-Proskauer (VP) test * These tests both use the same broth for bacterial growth. The broth is called MRVP broth. After growth, the broth is separated into two different tubes, one for the Methyl Red (MR) test and one for the Voges-Proskauer (VP) test.

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

IMViC Series Methyl Red Test 45

VP test The reagents used for the VP test are Barritt's A (alpha-napthol) and Barritt's B (potassium hydroxide). When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a pink-burgundy color (a positive VP test). This color may take 20 to 30 minutes to develop. E. coli does not produce acetyl methyl carbinol,

IMViC Series Voges-Proskauer Test  Leave uncapped for 15-20 minutes…….. MRVP broth 47

VP test .

Citrate Test This test uses Simmon's citrate agar to determine the ability of a microorganism to use citrate as its sole carbon source.

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

Citrate Utilization Test IMViC Series Citrate Utilization Test - + 51

IMViC tests These IMViC tests are useful for differentiating the family Enterobacteriaceae, especially when used alongside the Urease test. Except for the lowercase “i”, which is added for ease of pronunciation, each of the letters in “IMViC” stands for one of these tests. “I” is for indole; “M” is for methyl red; “V” is for Voges-Proskauer, and “C” is for citrate.

Triple sugar Iron Agar The tube is inoculated by stabbing into the agar butt (bottom of the tube) with an inoculating wire and then streaking the slant in a wavy pattern. Results are read at 18 to 24 hours of incubation.  

Triple sugar Iron Agar When there is a yellow butt and a red slant that indicates that the organism can ferment glucose. The change in color from red to yellow is due to the change in pH as lactic acid is produced. The pH indicator used is Phenol Red

Triple sugar Iron Agar Nonfermentative Gram-negative Bacilli Positive test

Triple Sugar Iron -/+/+ 56

Starch Agar Starch Agar is basically an all-purpose medium A plate of this medium is single streaked or spot inoculated with the organism to be tested. After incubation (preferably for several days) the medium is flooded with Gram's iodine The iodine reacts with starch to form a dark blue-colored complex. Any clear area around the growth of the culture after the addition of the iodine indicates the breakdown of starch by the organism due to its production of amylase, an extracellular enzyme.

Starch Agar and the Amylase Test Starch Agar and the Amylase Test The clear (non-blue) area around the culture on the left indicates the absence of intact starch – and therefore a positive reaction for the amylase test. The blue color extends up to the culture on the right which is thus seen to be unable to break down starch – a negative reaction for this test.

Skim Milk agar Skim Milk agar is used to detect the presence of caseinase. Caseinase is an enzyme that catalyzes the breakdown of casein into its individual amino acids.

Skim Milk agar Negative test

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

Catalase Catalase is an enzyme that converts hydrogen peroxide into water and oxygen. The presence of catalase can be easily detected by the slide method. A drop of 3 percent hydrogen peroxide is put on a slide and the bacteria is emulsified in the drop. The presence of bubbles is evidence of the production of oxygen

Catalase

What is the name of the lab procedure demonstrated here? Answer: Stabbing the butt! 64

C I M V U Answer: Escherichia coli Use the chart and/or the flow chart your lab manual to figure out who I am! Answer: Escherichia coli 65

Answer: Serratia marcesens V C U Use the and/or the flow chart on of your lab manual to figure out who I am! Answer: Serratia marcesens 66

Answer: Enterobacter aerogenes U 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 67