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The Staining Characterization of a Bacterial Unknown

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then use the information provided in figure 15.1 and table 15.1 to guide you through the characterization of your unknown. Perform only those staining procedures required as you work your way through the scheme. When you are finished, you should be able to correctly identify your bacterial unknown based on its staining characteristics.
Grampositive

Background
This exercise will allow you to apply what you have learned in previous exercises to the identification of a bacterial unknown. First, you will select or have assigned to you one of the bacterial cultures from the materials list, but you will not know which one. You will

Gram stain (Exercise 11) Gramnegative

Morphology (Exercises 9, 10, 11) Rod Capsule stain (Exercise 14) Coccus Neisseria sicca

Morphology (Exercises 9, 10, 11) Rod Acidfast stain (Exercise 12) Coccus Arrangement (Exercises 10, 11)

()

(+)

Clusters

Short chains

Alcaligenes denitrificans

Enterobacter aerogenes

Staphylococcus epidermidis

Enterococcus faecalis

()

(+)

Spore stain (Exercise 13) () (+)

Mycobacterium phlei*

Corynebacterium pseudodiphtheriticum

Bacillus cereus*

*45-day cultures will yield some Gram-negative cells.

Figure 15.1 Identification scheme for eight bacterial unknowns. You can use this
scheme when identifying your staining unknown. 101

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Table 15.1 Cell Size, Shape, and Arrangement of Bacterial Staining Unknowns
Bacterial culture
Alcaligenes denitrificans Bacillus cereus Corynebacterium pseudodiphtheriticum Enterobacter aerogenes Enterococcus faecalis Mycobacterium phlei Neisseria sicca Staphylococcus epidermidis

Cell size (m)

0.5!12 1!35 0.5!12 0.5!12 0.51 0.2!12 0.51 0.51.5

Cell shape
Rod Rod Rod Rod Coccus Rod Coccus Coccus

Cell arrangement
Single cells or pairs Streptobacilli Single cells or V-shapes Single cells or pairs Single cells, pairs, or short streptococci Single cells or cords Single cells or diplococci Single cells, pairs, or staphylococci

Materials
Cultures (1824-hour agar or broth) Alcaligenes denitrificans Bacillus cereus Corynebacterium pseudodiphtheriticum Enterobacter aerogenes Enterococcus faecalis Mycobacterium phlei Neisseria sicca Staphylococcus epidermidis
All agents in red are BSL2 bacteria.

Spore stain Malachite green Safranin Capsule stain Acidic stain: india ink Basic stain: crystal violet Equipment Hot plate (optional) Light microscope Miscellaneous supplies Bibulous paper Bunsen burner and striker Clothespin Disposable gloves (optional) Egg albumin solution Glass slides Immersion oil Inoculating loop or needle Lens paper Ocular micrometer Stage micrometer slide Staining tray Wash bottle with tap water Wax pencil

Stains Gram stain Crystal violet Grams iodine Ethanol (95%) Safranin Acid-fast stain Carbolfuchsin Acid-alcohol Methylene blue

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Procedure
1. You will select or have assigned to you an unknown from the materials list. Record your unknown number in the laboratory report. 2. Examine the information provided in figure 15.1 and table 15.1. Notice that the Gram stain must be done first to determine Gram reaction and cell morphology (figure 15.1). A negative stain or simple stain may be used in conjunction with a

Gram stain to verify cell size, shape, and arrangement (table 15.1). Notice that Grampositive and Gram-negative cocci will require no additional staining, but Gram-positive and Gramnegative rods will require one or more additional stains for identification. So, whatever your Gram stain results, continue to follow the identification scheme downward until you identify your unknown based on staining characteristics. 3. As you perform whatever stains are required, record your results in the laboratory report.

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L A B O R AT O RY R E P O RT
NAME LAB SECTION DATE

15

The Staining Characterization of a Bacterial Unknown

Unknown no. 1. Follow the information provided in figure 15.1 and table 15.1 to identify your staining unknown. Perform only the stains required to identify your unknown. 2. Required staining results: Gram stain (See Exercise 11.) Acid-fast stain (See Exercise 12.)

Magnification Cell size Cell shape Cell arrangement Cell color Gram reaction Magnification Cell shape Cell arrangement Cell color Acid-fast?

105

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Spore stain (See Exercise 13.)

Capsule stain (See Exercise 14.)

Magnification Cell shape Cell arrangement Vegetative cell color Endospores? If yes, color? If yes, location in cell Free spores? If yes, spore color? 3. Summary of the staining characteristics of my unknown:
Unknown no. Cell shape

Magnification Color of background Color of cells Cell shape Capsule? If yes, capsule color?

Cell arrangement

Gram reaction

Acid-fast?

Spores?

Capsules?

4. After examining the information provided in figure 15.1 and table 15.1 and recording the results of required stains, I conclude that my staining unknown is .

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5. Based on the information provided in figure 15.1 and table 15.1, fill in the following table for the eight unknown cultures.
Unknown culture Cell shape Cell arrangement Gram reaction Acidfast Spores Capsules

Alcaligenes denitrificans

Bacillus cereus

Corynebacterium pseudodiphtheriticum

Enterobacter aerogenes

Enterococcus faecalis

Mycobacterium phlei

Neisseria sicca

Staphylococcus epidermidis