MICROBIOLOGY LAB TESTS

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MICROBIOLOGY LAB TESTS
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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)

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

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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.

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Gram stainProcedure

• 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

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Procedure continue

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

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Gram Positive bacteria

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

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Gram Negative bacteria

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

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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.

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Motility

• Test Tube Results
• Test tube 1 Motile
• Test Tube 2Non-motile
• Test Tube 3Motile

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Motility Test
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EMB ( Eosin Methylene blue agar)

• Purpose This agar a is selective and
  differential medium used for isolation and
  differentiation among Enterobacteriaceae.

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EMB ( Eosin Methylene blue agar)

• EMB is selective for gram negative bacteria
  meaning only gram negative bacteria will grow on
  this medium.

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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

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EMB with E. coli
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Organisms which do ferment lactose will have pink
colonies.
                                                                                     

       
                                                                                                                                                

• .

                                                                     
                                                                                     

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Organisms which do ferment lactose will have pink
colonies
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MacConkey agar

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

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MacConkey agar

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

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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

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Organisms which do not ferment lactose will have
colorless colonies

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Organisms which do ferment lactose will have pink
colonies
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MacConkey Agar
Left Shigella - result Right
Escherichia result
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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

• Answers
• A 3. B
• 2. A 4. B

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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)

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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

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Carbohydrate fermentation ( glucose, lactose,
sucrose)
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Carbohydrate Fermentation Tests
Glucose w/ Durham Fermentation Tube
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Carbohydrate Fermentation Tests
Sucrose Fermentation
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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.

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Gelatine liquefaction test
                                     
                                                                           
                                                                           

• Positive Test

• Negative Test

                                           
                                                                                    



                                 

                             



                                     
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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

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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

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The Staphylococcus aureus ferments mannitol and
turns the medium yellow.  The Serratia marcescens
does not grow because of the high salt content
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Streptococcus agalactiae does not grow on MSA
because of the high salt content. Staphylococcus
epidermidis grows but does not ferment mannitol.
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Indol

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

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IndolWith the Kovacs reagent added

• Positive Test

• Negative Test

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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.

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SIM Test(, sulfur reduction portion)
                                 
                                                                    

                    
   

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

• No precipitate means a negative test

                              
                              

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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.

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These four tests include

• Indole test ( discussed earlier)
• Methyl Red test
• Voges-Proskauer (VP) test
• Citrate Test

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Indole Test
IMViC Series
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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.

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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.

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Methyl Red Test
IMViC Series
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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,

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Voges-Proskauer Test
IMViC Series
Leave uncapped for 15-20 minutes..
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VP test

• .

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Citrate Test

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

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Simmons Citrate Test

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

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Citrate Utilization Test
IMViC Series
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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.

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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.  

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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

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Triple sugar Iron Agar

• Nonfermentative Gram-negative Bacilli

• Positive test

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Triple Sugar Iron
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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.

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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.

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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.

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Skim Milk agar

• Negative test

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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

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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

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