Microbiology 101LABORATORY 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

Litmus milk reactions

SIM

Urease

Nitrate

Wear gloves!Let developer soak into platebefore discarding

Carbohydrates are the preferred carbon source because they are:

1. water soluble2. easily found in the environment3. easily oxidized and reduced to make energy4. non-toxic in dilute concentrations

Why carbohydrates?

• 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

Today’s Experiments:

If an ETC is not used, fermentationallows regeneration of e- carrier (NAD+)

Reduce pyruvate toLactate (lactic acid)

to regenerateNAD+

What is the terminal e- acceptor?

Fermentation products

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

RespirationProducts

-usually neutralor basic

Fermentations

F tubes (Durham tube)

PHENOL REDBroth BasePhenol Red Broth Base is a liquid medium to which carbohydrates may be 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 WaterTrypticase Peptone 10.000Sodium Chloride 5.000Phenol 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 organismdoesn’t grow, doesit mean they can’t ferment the sugar?

A. Fermentations

KIA reactionsSugar reversionProtein sparingMetabolic hierarchy

Organisms Slant Butt Gas Reaction (e.g. A/AG) Escherichia coliBacillus subtilisAlcaligenes faecalisEnterobacter aerogenesProteus vulgarisPseudomonas fluorescens Bacterial unknown

KIA

Phenol red (pH indicator)

below pH 6.8

above pH 8.2

6.8 ↔ 8.2

Should agree with F-tube results

Stab and streak

B. Specific end products of fermentations: mixed acid and 2, 3 butanediol

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 WaterPolypeptone Peptone 7.0Dextrose 5.0Potassium 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.

Methyl Red Voges-Proskauer (MRVP)

1. Growth2. Methyl Red3. Voges-Proskauer

Must incubate for 5-7 days to allow for complete conversion of end products

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

Table 1. pH indicators and their useful ranges of pH Color

Indicator pH range _________ _________________Acid Alkali

________________________________________________________________________________Thymol blue 8.0-9.6 Yellow BluePhenol red 6.8-8.4 Yellow RedBromothymol blue 6.0-7.6 Yellow BlueBromocresol purple 5.2-6.8 Yellow PurpleMethyl red 4.4-6.0 Red YellowBromocresol green 3.8-5.4 Yellow BlueBromophenol blue 3.0-4.6 Yellow Blue________________________________________________________________________

Methyl red

Add methyl red

Add KOH/Creatine& Alpha Naphthol

Voges-Proskauer

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

C. Hydrolysis of starch

Organisms Reaction to IodineB. subtilisE. coliBacterial unknown

Iodine + starch blueGrowth

D. Citrate utilization

Organisms Citrate utilizationE. aerogenesE. coliBacterial unknown

If bacteria use citrate as a Csource, they will grow. Ammonium phosphate willbe converted to ammonia and ammonium hydroxide increasing the pH

• 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.