Microbiology Laboratory Experiments Overview
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Professor Diane Hilker
I.
Exp. 15: Physiology of Bacteria
Purpose:
To examine specific enzymatic
activities of microbes that are frequently used
to identify bacterial species.
Refer to Exp. 21, Table 9 & Figure 8, for
biochemical reactions of various Gram neg.
rods
Inoculated last lab:
◦
Gram Neg. rods:
E.coli, Enterobacter,
Proteus
◦
Gram Pos. rod:
Bacillus stearothermophilus
Phenol Red Dextrose Broth (PRDB):
does the
microbe ferment glucose or dextrose?
◦
Yellow with gas: +
◦
Yellow without gas: -
◦
Red with or without gas: -
+ - -
◦
Yellow/red with or without gas: +/-
Phenol Red Lactose Broth (PRLB):
does the
microbe ferment lactose?
◦
Yellow with gas: +
◦
Yellow without gas: -
◦
Red with or without gas: -
+ - -
◦
Yellow/red with or without gas: +/-
Nitrate Broth:
Does the microbe produce an
enzyme called nitratase?
Nitrate
Nitrite
Nitratase
ADD:
2-3 drops Nitrate A
Mix; look for color
2-3 drops Nitrate B
development in 30sec.
Nitrate Broth
RESULTS:
Peach/pink: +
Not peach/pink: -
- +
Tryptone Broth:
Does the microbe produce
an enzyme called tryptophanase?
Tryptophan
Indole
Tryptophanase
ADD:
10-12 drops of Kovacs Reagent
Mix; look
immediately
for the reaction
Tryptone Broth
RESULTS:
Maroon top layer: +
No maroon top layer: -
Methyl Red- Voges Proskauer Broth (MRVP)
First
divide the tube in half using a Pasteur
pipette. Transfer ½ of broth to a 2
nd
empty
glass tube. Cap both tubes. One tube you will
perform the Methyl Red Test and the 2
nd
the
Voges Proskauer Test
Methyl Red Test:
Does the microbe produce
a large amount of acid end product from
glucose fermentation?
Glucose
pH below 4.4
ADD:
4 drops of Methyl Red Reagent
Mix; look
immediately
for the reaction
Methyl Red Test
RESULTS:
Pink: +
Not Pink: -
Voges Proskauer Test:
Does the microbe
produce a compound called acetoin during
glucose fermentation?
Glucose
Acetoin
ADD:
18 drops of Barritts A Reagent and
18 drops of Barritts B Reagent
Mix tube well
& let stand for 10 minutes. If neg.,
return tube to rack and look periodically over the
next 50 minutes for a pos. reaction.
Do not
reshake tube.
Voges Proskauer Test
RESULTS:
Top maroon/pink
film
: +
Film NOT maroon/pink: -
Simmon’s Citrate Test:
Does the microbe
utilize citrate as a source of carbon?
The Simmons Citrate slant contains citric
acid and bromothymol blue (green when
acidic). When citrate is utilized, the pH
increases to 7.6 and bromothymol blue
turns blue.
Simmon’s Citrate Test
RESULTS:
Observe for color change on the
surface
of the slant
Blue slant: +
Green slant: -
- +
Hydrolysis of Urea:
Does the microbe
produce an enyzme called urease that breaks
down urea?
Urea Broth tube contains urea and phenol
red. When urea is broken down to CO
2
&
ammonia, the pH increases to 8.4 and phenol
red turns to a magenta/purple color.
Urea
Ammonia (pH 8.4)
Hydrolysis of Urea
RESULTS
:
Observe for a color change
Magenta/purple: +
Not magenta/purple: -
- +
Production of Hydrogen Sulfide (H
2
S):
Does
the microbe produce H
2
S when decomposing
proteins?
TSI Tube
must be stabbed. The tube contains
three sugars and iron. Only analyzing the
tube for H
2
S production which is indicated by
a black coloration (any amount) in the butt or
bottom of the tube.
Hydrogen Sulfide (H
2
S) Test
RESULTS:
Black color in butt/bottom of tube: +
No back color in butt/bottom of tube: -
- - + -
Hydrolysis of Starch:
Does the microbe
produce an exoenzyme called amylase that
breaks starch into maltose?
amylase
Starch
Maltose
+ +
IKI IKI
Brown Clear
Hydrolysis of Starch
ADD:
Flood the plate with iodine or IKI.
Observe for a clearing around colony.
Clear halo surrounding colony: +
Brown color surronding colony: -
Professor Diane Hilker
I.
Exp. 16: Identification of Unknown Microorganisms
Purpose:
To determine the identity of
unknown microorganisms by using various
test methodologies.
TODAY:
◦
Set up Temperature Study (Exp. 7):
5, 25, 37, 60
o
C
◦
Set up Colony Morphology Plate
◦
Do a Wet Mount:
motility & size
◦
Do a Simple Stain:
cocci, rod or large ovoid cells
◦
Do a Gram Stain:
Gram + or -; shape
Gram Stain
Yeast
Gram Neg.Rod Gram Pos. Cocci
This overview showcases various microbiology laboratory experiments conducted to examine the physiology of bacteria, enzymatic activities, and biochemical reactions. It includes experiments on Phenol Red Dextrose Broth, Phenol Red Lactose Broth, Nitrate Broth, Tryptone Broth, and more. The provided images and descriptions help identify bacterial species based on activities of microbes. The experiments involve observing color changes, enzyme production, and gas formation, offering insights into microbial characteristics and behaviors.
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Presentation Transcript
Exp. 15: Physiology of Bacteria Exp. 15: Physiology of Bacteria I. I.
Purpose: activities of microbes that are frequently used to identify bacterial species. Purpose: To examine specific enzymatic Refer to Exp. 21, Table 9 & Figure 8, for biochemical reactions of various Gram neg. rods Inoculated last lab: Gram Neg. rods: E.coli, Enterobacter, Proteus Gram Pos. rod: Bacillus stearothermophilus
Phenol Red Dextrose Broth (PRDB): microbe ferment glucose or dextrose? Yellow with gas: + Phenol Red Dextrose Broth (PRDB): does the Yellow without gas: - Red with or without gas: - + + - - - - Yellow/red with or without gas: +/-
Phenol Red Lactose Broth (PRLB): microbe ferment lactose? Yellow with gas: + Phenol Red Lactose Broth (PRLB): does the Yellow without gas: - Red with or without gas: - + + - - - - Yellow/red with or without gas: +/-
Nitrate Broth: enzyme called nitratase? Nitrate Broth: Does the microbe produce an Nitrate Nitrite Nitratase ADD: ADD: 2-3 drops Nitrate A 2-3 drops Nitrate B development in 30sec. Mix; look for color
Nitrate Broth Nitrate Broth RESULTS: RESULTS: Peach/pink: + Not peach/pink: - - - + +
Tryptone an enzyme called tryptophanase? Tryptone Broth: Broth: Does the microbe produce Tryptophan Indole Tryptophanase ADD: ADD: 10-12 drops of Kovacs Reagent Mix; look immediately immediately for the reaction
Tryptone Tryptone Broth Broth RESULTS: RESULTS: Maroon top layer: + No maroon top layer: -
Methyl Red Methyl Red- - Voges Voges Proskauer Proskauer Broth (MRVP) Broth (MRVP) First pipette. Transfer of broth to a 2ndempty glass tube. Cap both tubes. One tube you will perform the Methyl Red Test and the 2ndthe Voges Proskauer Test First divide the tube in half using a Pasteur
Methyl Red Test: a large amount of acid end product from glucose fermentation? Methyl Red Test: Does the microbe produce Glucose pH below 4.4 ADD: ADD: 4 drops of Methyl Red Reagent Mix; look immediately immediately for the reaction
Methyl Red Test Methyl Red Test RESULTS: RESULTS: Pink: + Not Pink: -
Voges produce a compound called acetoin during glucose fermentation? Voges Proskauer Proskauer Test: Test: Does the microbe Glucose Acetoin ADD: ADD: 18 drops of Barritts A Reagent and 18 drops of Barritts B Reagent Mix tube well return tube to rack and look periodically over the next 50 minutes for a pos. reaction. Do not reshake Mix tube well & let stand for 10 minutes. If neg., Do not reshake tube. tube.
Voges Voges Proskauer Proskauer Test Test RESULTS: RESULTS: Top maroon/pink film Film NOT maroon/pink: - film: +
Simmons utilize citrate as a source of carbon? Simmon s Citrate Test: Citrate Test: Does the microbe The Simmons Citrate slant contains citric acid and bromothymol blue (green when acidic). When citrate is utilized, the pH increases to 7.6 and bromothymol blue turns blue.
Simmons Simmon s Citrate Test Citrate Test RESULTS: surface RESULTS: Observe for color change on the surface of the slant Blue slant: + Green slant: - - - + +
Hydrolysis of Urea: produce an enyzme called urease that breaks down urea? Hydrolysis of Urea: Does the microbe Urea Broth tube contains urea and phenol red. When urea is broken down to CO2& ammonia, the pH increases to 8.4 and phenol red turns to a magenta/purple color. Urea Ammonia (pH 8.4)
Hydrolysis of Urea Hydrolysis of Urea RESULTS: : Observe for a color change Magenta/purple: + Not magenta/purple: - RESULTS - - + +
Production of Hydrogen Sulfide (H the microbe produce H2S when decomposing proteins? Production of Hydrogen Sulfide (H2 2S): S): Does TSI Tube three sugars and iron. Only analyzing the tube for H2S production which is indicated by a black coloration (any amount) in the butt or bottom of the tube. TSI Tube must be stabbed. The tube contains
Hydrogen Sulfide (H Hydrogen Sulfide (H2 2S) Test S) Test RESULTS: RESULTS: Black color in butt/bottom of tube: + No back color in butt/bottom of tube: - - - - - + + - -
Hydrolysis of Starch: produce an exoenzyme called amylase that breaks starch into maltose? Hydrolysis of Starch: Does the microbe amylase Starch + + IKI IKI Maltose Brown Clear
Hydrolysis of Starch Hydrolysis of Starch ADD: ADD: Flood the plate with iodine or IKI. Observe for a clearing around colony. Clear halo surrounding colony: + Brown color surronding colony: -
Exp. 16: Identification of Unknown Microorganisms Exp. 16: Identification of Unknown Microorganisms I. I.
Purpose: unknown microorganisms by using various test methodologies. Purpose: To determine the identity of TODAY: Set up Temperature Study (Exp. 7): Set up Colony Morphology Plate Do a Wet Mount: Do a Simple Stain: Do a Gram Stain: TODAY: Set up Temperature Study (Exp. 7): 5, 25, 37, 60oC Set up Colony Morphology Plate Do a Wet Mount: motility & size Do a Simple Stain: cocci, rod or large ovoid cells Do a Gram Stain: Gram + or -; shape
Gram Stain Yeast Gram Neg.Rod Gram Pos. Cocci
