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Appendix B – Identifying Bacteria

Bacteria identification is accomplished in a number of ways.
Two common tools microbiologists use to identify unknown bacteria include
dichotomous key and biochemical tests. The dichotomous key is useful when a
microbiologist only needs to know which group an unknown microbe belongs to on
a general level. When a microbiologist needs to identify a specific bacterium,
biochemical tests are used.
PART ONE: GENERAL BACTERIA IDENTIFICATION

Review the dichotomous key in Figure A, the bacterial shapes
in Figure B, and the Gram stain information below. You will use all three to
determine to which major group unknown bacteria belong.

Figure A: A dichotomous key for classifying major groups of
bacteria.
[Figure 9.4 in Microbiology text]

Figure B: The most common bacterial shapes.
[Figure 4.1 in Microbiology text]

Shape Types:

Comma, or Club-shaped, rods = Vibrio
Rods = Coccobacillus, Bacillus
Spherical = Coccus
Spiral or Helical = Spirillium, Spirochete

Gram Stain Results:

Purple = Gram positive
Red = Gram negative
Neither purple nor red = No cell wall (neither Gram positive
nor Gram negative)

Review Figure C on the following page. Use Figure C to
identify the type of arrangement displayed by the unknown bacteria. Read
through the Sample Identification on the following page for an example of how
to identify bacterial groups and arrangements using Figures A, B, C, and the
Gram stain results.
Figure C: Arrangement of Bacteria
Rods and Clubs

Single

Strepto-

Cording

Snapping

Palisades

Chinese Letters

rod_arrangements
Cocci

Single

Diplo-

Tetrad

Sarcinae

Staphylo-

Strepto-

cocci_arrangements
Sample Identification

After performing a Gram stain, you observe the following
under a microscope:

gram_negative_cocci
(Cells are stained red.)

· Begin with
item 1 on the dichotomous key (Figure A). Because the organisms are red, they
are not Gram-positive. According to the key, continue to item 3.
· Because the
organism is red (not clear or another color), it is indeed Gram-negative
according to item 3. The key directs you to item 5.
· Consider the
shape of individual cells for item 5. According to Figure B, the shape type is
closest to spherical. (Note: spherical is circular.)
· The bacteria
belong to the Gram-negative cocci group.
· Now use
Figure C to describe arrangement of bacteria. (The bacteria are not rod- or
club-shaped, so focus on the cocci arrangements.) Identify the arrangement that
is most prominent.
· The bacteria
are arranged in a diplo- fashion.
· Enter the
group and arrangement in the table.

Application

Use Figures A, B, C, and Gram stain results to identify
group and arrangement of bacteria. Continue to Part Two after completing the
table.

View from Microscope
Group of Bacteria
Arrangement
gram_negative_cocci
(stained red)
Gram-negative cocci
Diplo-
gram_positive_bacilli
(stained purple)

gram_positive_cocci
(stained purple)

vibroids
(stained red)

spirochete
(stained red)

corynebacteria
(stained purple)

gram_positive_cocci_staphylo
(stained purple)

PART TWO: CAse study scenario: identify specific bacteria
through biochemical testing

To gain an understanding of the processes involved with
identifying bacteria through biochemical tests, access Chapter 6 of the text in
WileyPlus located on the Week One course page. Once in WileyPlus (Chapter 6),
select the “Bacterial Identification by API” link located under the heading,
Take Another Look. Once selected, review the information and watch the
Flash-animated movie (animation) located within this link. Then, review the
following scenario and answer the questions that follow.
Case Study

A recent outbreak of food poisoning has occurred in a
community. One possible source of contamination may be the produce that is
grown and distributed locally at a farmer’s market. A test sample of some of
the produce revealed evidence of bacterial contamination. The bacteria sample
was tested in a microbiology lab and showed the results that follow. The
laboratory also performed a Gram stain of the isolated bacteria and ran a
number of biochemical tests to aid identification. The biochemical tests were
assayed using the Analytical Profile Index (API) 20E system for identification
of Enterobacteriaceae and other gram-negative bacteria.

Figure 1. Microbiology laboratory results – Unknown bacteria
present on produce (API Results)

entero1entero2

Figure 2. API Results of Unknown Bacteria in Text Format

Gram stain:
Gram-negative

ONPG

ADH

LDC

ODC

CIT

H2S

URE

TDA

IND

VP

GEL

GLU

Bacteria: Unknown
+

+

+

+

+

MAN

INO

SOR

RHA

SAC

MEL

AMY

ARA

+

+

+

+

+

Based on Gram stain results and the knowledge that this
bacterium caused food poisoning, the laboratory is able to narrow down the
possibilities to three bacterial strains. Figure 3 shows the API results:

Figure 3. API Results of Known Bacterial Strains in Text
Format

Gram stain:
Gram-negative

ONPG

ADH

LDC

ODC

CIT

H2S

URE

TDA

IND

VP

GEL

GLU

Bacteria: Salmonella

+

+

MAN

INO

SOR

RHA

SAC

MEL

AMY

ARA

+

+

+

Gram stain:
Gram-negative

ONPG

ADH

LDC

ODC

CIT

H2S

URE

TDA

IND

VP

GEL

GLU

Bacteria: Shigella

+

MAN

INO

SOR

RHA

SAC

MEL

AMY

ARA

Gram stain:
Gram-negative

ONPG

ADH

LDC

ODC

CIT

H2S

URE

TDA

IND

VP

GEL

GLU

Bacteria: E. coli
+

+

+

+

+

MAN

INO

SOR

RHA

SAC

MEL

AMY

ARA

+

+

+

+

+

Application
Compare the API biochemical test results in Figure 2
(unknown bacteria from produce) with API test results in Figure 3 (known
bacterial strains identified in the gray boxes on the left). The bacterial
strain in Figure 3 that matches Figure 2 will identify the bacterial strain
causing illness. Based upon your observations, which of the bacteria in Figure
3 is the most likely cause of the food poisoning outbreak?

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