Microbiology 101

LABORATORY EXERCISE #22:

Carbohydrate metabolism
Today in Lab:
• Check your Nitrogen Metabolism results
• Perform Carbohydrate Metabolism inoculations
• No differential media work
• No PCR progress, more on that next lab
LABORATORY EXERCISE #21: Nitrogen metabolism

Gelatin
hydrolysis Wear gloves!
Let developer soak into plate
before discarding

Litmus milk
reactions

SIM Nitrate

Urease
 Why carbohydrates?

Carbohydrates are the preferred carbon

source because they are:
1. water soluble
2. easily found in the environment
3. easily oxidized and reduced to make energy
4. non-toxic in dilute concentrations
 Today’s Experiments:
• Fermentation of different carbohydrates
– F tubes (aka Durham tubes)
– Kligler Iron Agar (KIA)
• Detection of different fermentation
products
– MRVP
• Hydrolysis of starch
– Starch agar
• Utilization of citrate as a sole carbon source
– Simmon’s Citrate Agar
 If an ETC is not used, fermentation
allows regeneration of e- carrier (NAD+)

Reduce pyruvate to
Lactate (lactic acid)
to regenerate
NAD+

What is the terminal e- acceptor?

 Fermentation
products
-acids (ate/ic)
-alcohols
-gas (CO2)

Respiration
Products
-usually neutral
or basic
 PHENOL RED
Fermentations Broth Base
Phenol Red Broth Base is a liquid medium to which carbohydrates may be
F tubes (Durham tube) added for accurate determination of fermentation reaction. Its basic
constituent is casein peptone, previously tested for freedom from
fermentable carbohydrate.
______________________________________________________
Formula in Grams per Liter of Distilled Water
Trypticase Peptone 10.000
Sodium Chloride 5.000
Phenol Red 0.018
Final pH 7.4+
________________________________________________
Preparation. Dissolve 15 grams in a liter of distilled water. If desired,
carbohydrate (five to ten grams) may be added. Agar (0.5 to 1.0 grams)
may also be added if a fluid medium is desired for cultivation of anaerobic
organisms. Durham tubes may be used for detection of gas formation.
Arrange tubes loosely in suitable containers and sterilize at 116Á to
188ÁC. (not over 12 lbs. steam pressure) for 15 minutes.

If the organism
doesn’t grow, does
it mean they can’t
ferment the sugar?
A. Fermentations
KIA reactions
Sugar reversion
Protein sparing
Metabolic hierarchy
Stab and streak KIA

Phenol red (pH indicator)

below pH above pH
6.8 8.2
6.8 ↔ 8.2

Organisms Slant Butt Gas Reaction (e.g.

A/AG) Escherichia coli
Bacillus subtilis
Alcaligenes faecalis Should agree
Enterobacter aerogenes
Proteus vulgaris with F-tube
Pseudomonas fluorescens
Bacterial unknown
results
B. Specific end products of fermentations: mixed acid and 2, 3 butanediol
Methyl Red Voges-Proskauer (MRVP)
MR-VP BROTH (Clark and Lubs Medium)
MR-VP Broth is used in differentiation of bacteria by means of the Methyl
Red and Voges-Proskauer reactions. It is recommended for use in
differentiation of coliform organisms according to the American Public
Health Association's Standard Methods for the Examination of Dairy
Products, Water and Wastewater.
______________________________________________________
Formula in Grams per Liter of Distilled Water
Polypeptone Peptone 7.0
Dextrose 5.0
Potassium Phosphate 5.0
Final pH 6.9±
______________________________________________________
Preparation. Suspend 17 grams of the powder in a liter of distilled water.
Mix thoroughly. If necessary, heat slightly to dissolve. Dispense and
sterilize at 118¡ to 112¡C (not over 15 lbs. pressure) for 15 minutes.

1. Growth
2. Methyl Red
3. Voges-Proskauer

Must incubate for 5-7 days to allow for

complete conversion of end products
Methyl red
Mixed acid (E. coli):
Glucose (6-C)-->2-Pyruvate (3-C) + 2ATP
-->50% Lactic acid (3-C)
-->50% ‘Mixed acids’
Formic (1-C) + CO2/Acetic (2-C)/Ethanol (2-C) + CO2

2,3 Butanediol (Enterobacter aerogenes):

Glucose (6-C)-->2-Pyruvate (3-C) + 2ATP
-->Lactic acid(small amount)
--> CO2 + H2O
--> Acetic-->2-3 butanediol
->Mostly alcohols/gas (neutral end products)
 Methyl red

Add methyl red

Table 1. pH indicators and their useful ranges of pH
Color
Indicator pH range _________ _________________
Acid Alkali
________________________________________________________________________
________
Thymol blue 8.0-9.6 Yellow Blue
Phenol red 6.8-8.4 Yellow Red
Bromothymol blue 6.0-7.6 Yellow Blue
Bromocresol purple 5.2-6.8 Yellow Purple
Methyl red 4.4-6.0 Red Yellow
Bromocresol green 3.8-5.4 Yellow Blue
Bromophenol blue 3.0-4.6 Yellow Blue
________________________________________________________________________
Voges-Proskauer
Add KOH/Creatine
& Alpha Naphthol

You must do the MR test first! Why???

C. Hydrolysis of starch

Organisms Reaction to
Iodine
B. subtilis
E. coli
Bacterial unknown

Growth Iodine + starch  blue

D. Citrate utilization
If bacteria use citrate as a C
source, they will grow.
Ammonium phosphate will
be converted to ammonia and
ammonium hydroxide
increasing the pH

Organisms Citrate
utilization
E. aerogenes
E. coli
Bacterial unknown
• Know the reason for including each component of the media used
this lab.
• Know the information derived from each medium.
• Understand the importance of using proper controls.
• Determine the information derived from each medium in terms of
the characteristics of your unknown culture.
• Explain the carbohydrate metabolism for each of microbes presented
in today’s lab.