OBSERVING  METABOLIC ACTIVITY

OF MICROORGANISMS

Cultures of Citrobacter freundii, Staphylococcus epidermidis, Serratia marcescens, Bacillus subtilis, Proteus vulgaris, Escherichia coli and Enterbacter aerogenes will be used for each lab section.

STUDENT INSTRUCTIONS

     Many species of organisms are identical with respect to morphology and staining patterns.  In order to differentiate them, characteristics of metabolism must be observed.  There are many types of medium specifically designed to feature differences in metabolic pathways.  You will inoculate several examples today.

     Working in teams of four, inoculate an example of each media tube provided with one test organism.
      Descriptions and inoculation instructions for each media type are described as follows:

I.  Phenol red carbohydrate broths

Phenol red carbohydrate broths are used in fermentation studies of pure cultures for cultural identification of microorganisms.  They consist of a complete basic medium prepared with phenol red as an indicator of changes in pH; carbohydrates are added according to designs of the experiment.  Each tube will have a single carbon/energy source.  If the organism possesses pathways of fermentation, acid is a likely product, changing the medium from red to yellow.  When used with a Durham tube, gas production as a result of fermentation is also shown.   A bubble will appear in the inverted tube following incubation.

          Inoculate the three tubes provided, incubate 48 hours, observe and record the results:

      Key to symbols and abbreviations:
                  +  =  positive for fermentation of the sugar
                  -   =  negative for fermentation of the sugar
                  A  =  positive for acid production (red turn to yellow)
                  G  =  positive for gas production (bubble in Durham tube)
                 OA  = no acid
                 OG  = no gas

II. Simmon’s Citrate Agar

In Simmon's citrate agar, citrate is the only carbon/energy source.  If the organism can utilize citrate for energy, it must deaminate it, releasing alkaline products.  The pH indicator is bromythymol blue, which appears green in the neutral state.  A positive reaction to Simmon’s citrate medium is a change from green to royal blue.  Stab inoculate as demonstrated by the instructor to allow growth throughout the medium.  (Introduce the inoculation loop below the agar surface and stab to the bottom of the tube, withdrawing gently along the stab line).

Some organisms possess the enzyme gelatinase.  When stab-inoculated into nutrient gelatin medium, they will liquefy the gelatin: i.e. it will not resolidify even after refrigeration.  Stab inoculate a tube of nutrient gelatin with your test organism, incubate 48 hours, refrigerate 30-60 minutes before observation, then observe and record results.  If the medium does not resolidify, the organism is considered positive for gelatinase.
 

Organism:	Results: (+ or -)

 

A tube of urea broth or agar has urea as the only carbon/energy source.  In order to ferment urea, the organism must possess the enzyme urease.  This enzyme breaks down urea, releasing the alkaline amino group.  The pH indicator phenol red goes to the alkaline “hot pink” color.  Inoculate as normal for broth; or stab inoculate for urea agar.  Incubate 48 hours or longer, observe and record results.

 V. Triple Sugar Iron (TSI) Agar

Triple Sugar Iron (TSI) agar is characterized by a short slant and a deep butt.  The slant should be stroked and then the butt stabbed.  There are lactose, sucrose, and glucose present in a 10:10:1 ratio.  If the organism can ferment either of the disaccharides, the entire tube will show yellow (phenol red is the pH indicator) after 24 hours.  If glucose only is fermented, the organism goes to protein in the agar as a nutrient source following exhaustion of the glucose supply.  Alkalis are produced and the indicator at the slant goes to pink by 48 hours.  (The butt remains yellow due to the lower oxygen tension in this area of the tube).

If gas is produced from any carbohydrate fermentation, the agar will be split.  (Since the color changes can indicate a difference between monosaccharide and disaccharide use but not between lactose and sucrose use, the TSI media has been replaced in many labs by Kligler’s sugar and iron agar in order to avoid confusion between the two disaccharides.  Since many organisms were first identified by their reactions on standardized tests including TSI, and are still so described in many texts, we will use this historically significant medium for our studies).

Ferrous sulfate in the medium will react with any hydrogen sulfide produced to form ferrous sulfide, a black precipitate.

TSI agar provides a means of confirmation of the carbohydrate broth test and allows evaluation of another metabolic feature, the production of H₂S.

Stroke the slant and stab inoculate the butt of the TSI medium with your test organism.  Incubate 48 hours, observe and record the results.
 

TSI REACTION:	EXPLANATION:
Acid butt (yellow) and  Alkaline slant (red or pink)               [written a/alk or y/p]	Glucose fermented, neither disaccharide fermented
Acid throughout  (yellow butt and slant)               [written a/a or y/y]	Either or both disaccharides fermented
Alkaline throughout (pink butt and slant)               [written alk/alk or p/p]	No sugars were fermented
Gas bubbles in butt, medium sometimes split               [add +G to notations above]	Positive for gas production
No gas bubbles or split agar               [add 0G to notations above]	Negative for gas production
Blackening of butt               [write +H2S]	Positive for hydrogen sulfide production

     Bergey’s Manual of Determinative Bacteriology has been a standard reference volume and has guided many students and professional microbiologists through identification procedures.

     It is helpful to work through identification questions in the form of a “flow-chart” in which the answer to one question determines the next question to be asked and answered, following a plan of elimination until the specific level of identification is reached.  Years of analysis and testing of known organisms in pure culture has provided the basis of comparison information used when searching for the identity of an unknown organism.

     For example: The Gram stain will divide bacteria into two groups, G+ and G-.  Then look for presence or absence of spores, shape (round, rod, or spiral)  and other staining properties (ex. acid-fast reaction). Check for motility, and then reactions on various metabolic media.  The following outline demonstrates an appropriate train of thought, flowing  from one possibility to another when trying to identify an organism.
 

I.  GRAM POSITIVE	II.  GRAM NEGATIVE
A. Rods:	A. Rods
1.  Spore-formers:	1.  Aerobes (non-fermentive)
a.  Bacillus:  strict aerobe	a.  Pseudomonas
b.  Clostridium:  strict anaerobe	b.  Alcaligenes
c. Sporolactobacillus:  microaerophilic
2.  Non-Sporeformers	2. Facultative Anaerobes (fermentive)
a.  Acid-fast:  Mycobacterium	a. Motile
	(1) Citrate Positive
	a) Lactose Positive
	i. Methyl Red Positive:Citrobacter
	ii. Methyl Red Negative:  Enterobacter
	b) Lactose Negative(MR+):Salmonella
	(2) Citrate Negative
	a) Lactose Positive (Urease-): Escherichia
	b) LactoseNegative(Urease+):Proteus
b.  Not Acid-fast	b. Non-Motile
(1) Lactobacillus	(1) Citrate Positive: Klebsiella
(2) Corynebacterium	(2) Citrate Negative: Shigella
(3) Propionibacterium
B.  Cocci	B. Cocci
1. Irregular and Tetrad Arrangement	1. Neisseria
a. Micrococcus
b. Staphylococcus
c. Planococcus
2. Pairs of Chains: Streptococcus	2. Veillonella