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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.