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Chapter 3.2 : Staining Procedures

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Abstract:

The Gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and Gram reactions; it is additionally a critical test for the presumptive diagnosis of infectious agents and serves to assess the quality of clinical specimens ( ). The test was originally developed by Christian Gram in 1884. The modification currently used for general bacteriology was developed by Hucker in 1921; it provides greater reagent stability and better differentiation of organisms. Other modifications have been specifically developed for staining anaerobes (Kopeloff's modification) and for weakly staining gram-negative organisms ( spp., spp., spp., etc.) by using a carbol fuchsin or basic fuchsin counterstain ( ). In fact, many laboratories use these counterstains routinely, especially for direct smears of clinical material.

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
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Figure 3.2.1-1

Thin-smear preparation.

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
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Figure 3.2.1-2

Touch preparation of smear.

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
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Figure 3.2.1-3

Typical Gram stain morphologies of gram-positive and gram-variable genera.

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
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Figure 3.2.1-4

Typical Gram stain morphologies of gram-negative genera.

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
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References

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50. Hoff, R. G.,, D. E. Newman,, and J. L. Stanek. 1985. Bacteriuria screening by use of acridine orange-stained smears. J Clin. Microbiol. 21: 513 516.
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53. Larson, A. M.,, M. J. Dougherty,, D. J. Nowowiejski,, D. F. Welch,, G. M. Matar,, B. Swaminathan,, and M. B. Coyle. 1994. Detection of Bartonella (Rochalimaea) quintanaby routine acridine orange staining of broth blood cultures. J. Clin. Microbiol. 32: 1492 1496.
54. Lauer, B. A.,, L. B. Reller,, and S. Mirrett. 1981. Comparison of acridine orange and Gram stains for detection of microorganisms in cerebrospinal fluid and other clinical specimens. J. Clin. Microbiol. 14: 201 205.
55. Lauer, B. A.,, L. B. Reller,, and S. Mirrett. 1981. Comparison of acridine orange and Gram stains for detection of microorganisms in cerebrospinal fluid and other clinical specimens. J. Clin. Microbiol. 14: 201 205.
1. Beverly, A. L.,, M. Venglarik,, B. Cotton,, and J. R. Schwebke. 1999. Viability of Trichomonas vaginalis in transport medium. J. Clin. Microbiol. 37: 3749 3750.
2. Borchardt, K. A.,, M. Z. Zhang,, H. Shing,, and K. Flink. 1997. A comparison of the sensitivity of the InPouch TM TV, Diamond's, and Tichosel media for detection of Trichomonas vaginalis. Genitourin. Med. 73: 297 298.
3. Fine, K. D.,, F. Ogunji,, J. George,, M. D. Niehaus,, and R. L. Guerrant. 1998. Utility of a rapid fecal latex agglutination test detecting the neutrophil protein, lactoferrin, for diagnosing inflammatory causes of chronic diarrhea. Am. J. Gastroenterol. 93: 1300 1305.
4. Guerrant, R. L.,, V. Araujo,, E. Soares,, K. Kotoff,, A. A. M. Lima,, W. H. Cooper,, and A. G. Lee. 1992. Measurement of fecal lactoferrin as a marker for fecal leukocytes. J. Clin. Microbiol. 30: 1238 1242.
5. Huicho, L.,, V. Garaycochea,, N. Uchima,, R. Zerpa,, and R. L. Guerrant. 1997. Fecal lactoferrin, fecal leukocytes and occult blood in the diagnostic approach to childhood invasive diarrhea. Pediatr. Infect. Dis. J. 16: 644 647.
6. Iida, T.,, A. Naka,, O. Suthienkul,, Y. Sakaue,, R. L. Guerrant,, and T. Honda. 1997. Measurement of fecal lactoferrin for rapid diagnosis of enterohemorrhagic Escherichia coli infection. Clin. Infect. Dis. 25: 167.
7. Lawing, L. F.,, S. R. Hedges,, and J. R. Schwebke. 2000. Detection of trichomonosis in vaginal and urine specimens from women by culture and PCR. J. Clin. Microbiol. 38: 3585 3588.
8. Lin, D. S.,, F. Y. Huang,, N. C. Chiu,, H. A. Koa,, H. Y. Hung,, C. H. Hsu,, W. S. Hsieh,, and D. I. Yang. 2000. Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants. Pediatr. Infect. Dis. J. 19: 223 227.
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1. Baron, E. J.,, G. H. Cassell,, D. A. Eschenbach,, J. R. Greenwood,, S. M. Harvey,, N. E. Madinger,, E. M. Paterson,, and K. B. Waites,. 1993. Cumitech 17A, Laboratory Diagnosis of Female Genital Tract Infections. Coordinating ed., E. J. Baron. American Society for Microbiology, Washington, DC.
2. Chapin, K., 2007. Principles of stains and media, p. 182 191. In P. R. Murray,, E. J. Baron,, J. H. Jorgensen,, M. L. Landry,, and M. A. Pfaller (ed.), Manual of Clinical Microbiology, 9th ed. ASM Press, Washington, DC.
3. Holmes, K. K.,, P. A. Mardh,, P. F. Sparling,, P. J. Wiesner,, W. Cates, Jr.,, S. M. Lemon,, and W. E. Stamm. 1990. Sexually Transmitted Diseases, 2nd ed. McGraw Hill Book Co., New York, NY.
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Tables

Generic image for table
Table 3.2.1-1

Gram stain modifications, recommended reagents, timing, and uses

Or, preferably, use 0.1 to 0.2% basic fuchsin as a counterstain ( ).

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
Generic image for table
Table 3.2.1-2a

Gram-positive organisms found in direct smears from some clinical sources

See references to and .

CYE, charcoal-yeast extract agar.

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
Generic image for table
Table 3.2.1-2b

Gram-positive organisms found in direct smears from some clinical sources

See references to and .

CYE, charcoal-yeast extract agar.

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
Generic image for table
Table 3.2.1-3a

Gram-negative organisms seen in direct smears from some clinical sources

See references to .

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
Generic image for table
Table 3.2.1-3b

Gram-negative organisms seen in direct smears from some clinical sources

See references to .

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
Generic image for table
Table 3.2.1-4

Common descriptions of bacterial Gram staining characteristics

Swelling of sides can suggest the presence of spores but may also be due to the presence of vacuoles, inclusions, marked pleomorphism, or irregular staining. Phase-contrast microscopy or a spore stain may be helpful in observing bacterial endospores.

The descriptive term “diphtheroid” or “coryneform” is used to describe gram-positive bacteria that are pleomorphic, club shaped, or irregularly staining or that have palisading and/or angular arrangements (V and L shapes).

Sizes listed are only guidelines. The average size of an RBC is 7 µm, and cytoplasmic granules in neutrophils average 0.2 to 0.3 µm.

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
Generic image for table
Table 3.2.1-5

Reporting Gram stain results

Determine the number of cells and bacteria in 20 to 40 fields of the smear using an 18- to 22-mm lens field of view. Skip fields where there are no cells or bacteria, and do not average these fields in the counts if there are fields where cells and/or bacteria are present.

The Gram stain is not a stain that demonstrates host cellular morphology, and the main cells routinely enumerated are WBCs (i.e., polymorphonuclear cells), which determine if the sample is purulent as an indication of infection. For other types of cells, there is no clinically useful reason to report cells present in amounts of <10/40 fields from a Gram stain. LPF, low-power field.

Ignore one or two microorganisms on the entire slide, unless the results can be reproduced on a second smear and only then if it is from an invasively collected specimen. OIF, oil immersion field.

Microbiologists should be encouraged to specify Gram stain morphology and resemblance to organism group consistent with the source if the presentation is classic; however, use caution when in doubt, as other species may mimic the typical presentations, including (i) gram-positive cocci in pairs, consistent with ; (ii) small gram-negative coccobacilli, consistent with ; (iii) tiny gram-positive bacilli, consistent with ; and (iv) gram-negative diplococci, consistent with .

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
Generic image for table
Table 3.2.1-A1

Standardized scoring method for evaluation of Gram stains for BV

Modified from Nugent et al. ( ) using quantitation from the Gram stain procedure. Circle in each row the number that corresponds to the quantitation visualized in the smear. Add circled numbers to arrive at total score. Interpret as follows: 0 to 3, normal; 4 to 6, intermediate; and 7 to 10, BV.

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2
Generic image for table
TABLE 3.2.3-1

Outline for reporting wet mount results from different specimens

Report the number of WBCs and RBCs per high-power field (HPF). Average several fields and report as follows: (i) heavy = ≥5/HPF, (ii) moderate = 1 to 4/HPF, (iii) few = < 1/HPF, and (iv) none = 0/HPF.

If the vaginal specimen is negative for parasites and was not preserved, or was not processed within 15 min, report “Unable to determine the presence of in unpreserved specimen or because of delay in transport.”

Citation: Garcia L. 2010. Staining Procedures, p 91-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.2

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