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BCH1200 Microbiology Lab - Manual PDF
BCH1200 Microbiology Lab - Manual PDF
BCH1200 Microbiology Lab - Manual PDF
Practical 1
Microbiology Lab: Identification of Bacteria
All bacterial cells have cell walls. This cell wall structure falls into two general types, which is
used as the major criterion for distinguishing different groups of bacteria. By staining the
bacteria with “Gram stain”, bacteria with a thick wall layer and do not have an outer membrane
can stain purplish blue and are called “Gram positive” (see Fig 1b). Bacteria with a thin cell
wall layer and an outer membrane do not retain the Gram stain after rinsing with alcohol but can
take up a red stain subsequently are called “Gram negative” (see Fig 1a).
Figure 1a. Gram stain of Gram-positive Figure 1b. Gram stain of Gram-negative
bacteria bacteria
(http://filebox.vt.edu/users/chagedor/biol_4684/Methods/cellwalls.html )
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Biochemical features of bacteria
Besides the cell wall structure, biochemical features of bacteria are usually used for
identification purpose. One of the examples is the presence or absence of oxidase, an enzyme
used to catalyze oxygen (O2) to water (H2O) or hydrogen peroxide (H2O2). This feature can be
identified using the “oxidase test”. Bacteria with the presence of oxidase can turn the oxidase
test reagent into dark purple are referred to as “oxidase positive” while the bacteria without
oxidase fail to turn the oxidase test reagent into purple (remain colorless) are referred to as
“oxidase negative”.
Different functional groups of bacteria have different nutritional requirements. They grow best
when specific types and optimal amount of sugars (e.g., glucose, adonitol, lactose, arabinose,
sorbitol, dulcitol), amino acids (e.g., lysine, ornithine, phenylalanine, tryptophan), peptides and
vitamins are provided. All these properties can be used for their identification based on their
fermentation (use) of these sugars and amino acids. These reactions will be illustrated in detail in
this practical.
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Principle of EnteroPluri-Test system
EnteroPluri-Test system (see Fig. 2) is a self-contained, compartmented plastic tube containing
12 different media that allows for the determination of 15 biochemical reactions.
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produce H2S (hydrogen sulfide), hydrolyze urea (produce ammonia) and/or to utilize citrate
as a carbon source.
The resulting combination of the 15 biochemical reactions together with the Interpretation Guide
(codebook), allow identification of clinically significant Enterobacteriaceae.
Materials Provided:
Four unknown bacteria, isolated from toilet or food samples, labeled A, B, C or D are
provided for identification.
Filter papers (Whatman No.1 or equivalent)
Sterilized toothpick
Oxidase test Reagent Droppers
Inoculated EnteroPluri-Test systems
Indole test Reagent Droppers (modified Kovacs' reagent)
Fine-point forceps
Color chart
Data record sheet
Interpretation Guide (Codebook)
Procedure:
2. One day before this practical, each EnteroPluri-Test system has been pre-inoculated with one
unknown bacteria by a technical officer using an inoculating wire. This allows inoculation of
all compartments in one step from one or a few single colonies of an isolate (see video
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demonstration). This pre-inoculation allows the bacteria to grow for one day and hence
sufficient time for the biochemical reactions to develop during their growth (Table 1).
4. Indole test
You still need to perform the final biochemical test, Indole test in the inoculated EnteroPluri-
Test system. It is a presumptive test to check whether the bacteria can utilize tryptophan (an
amino acid) to produce Indole. The procedures are shown as below:
a. Break the thin Mylar plastic film that covers the flat surface of the H2S/indole
compartment of EnteroPluri-Test system with a syringe or a fine-point forceps.
b. Inject 1 or 2 drops of Indole test reagent onto the surface of the medium in the
H2S/indole compartment.
c. Positive reaction – the reagent added turns red within 10 seconds.
Negative reaction – the reagent added remains colorless.
5. Reading results
a. After the tests, check positive test results for each EnteroPluri-Test system according to
the Color Chart given in Fig 3.
Negative
test result
Positive
test result
Figure 3. Color differences between uninoculated (negative) and inoculated (positive) tests.
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b. Record and circle the number below those tests of positive results on the Data Record
Sheet (see Fig. 4) provided.
Sample A
Example:
Sample A
Add up the
numbers of each
bracketed series
3 4 3 6 3
The “ID Value” 34363 can be found by thumbing the pages of the Interpretation
Guide provided. The listing is as follows:
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Table 1. Biochemical reactions measured by the EnteroPluri-Test system
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Table 1 (continued).