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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Acknowledgement
 ~  References

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Year : 2003  |  Volume : 21  |  Issue : 2  |  Page : 121-123
 

Vancomycin sensitivity and koh string test as an alternative to gram staining of bacteria


Department of Microbiology, PSG Institute of Medical Sciences and Research, Coimbatore - 641 004, Tamil Nadu, India

Correspondence Address:
Department of Microbiology, PSG Institute of Medical Sciences and Research, Coimbatore - 641 004, Tamil Nadu, India

 ~ Abstract 

Two hundred and eighteen bacterial isolates obtained from various clinical samples were subjected to Gram stain by conventional method. For all the isolates potassium hydroxide (KOH) string test and sensitivity to vancomycin were done. Gram positive bacteria showed 100% sensitivity to vancomycin as also 100% negativity for string test. Of the gram negative bacteria, 99.42% were resistant to vancomycin while 98.85% were positive for the string test. KOH and vancomycin tests are simple, inexpensive and can be used in addition to Gram staining for rapid identification of bacterial cultures.

How to cite this article:
Arthi K, Appalaraju B, Parvathi S. Vancomycin sensitivity and koh string test as an alternative to gram staining of bacteria. Indian J Med Microbiol 2003;21:121-3


How to cite this URL:
Arthi K, Appalaraju B, Parvathi S. Vancomycin sensitivity and koh string test as an alternative to gram staining of bacteria. Indian J Med Microbiol [serial online] 2003 [cited 2019 Aug 26];21:121-3. Available from: http://www.ijmm.org/text.asp?2003/21/2/121/7989


The initial classification of an unknown bacterium and subsequent identification procedures are largely dependent on the results of Gram stain. Based on this, bacteria are differentiated as gram positive and gram negative. The major pitfall in the Gram stain is the tendency of some gram positive bacteria to decolourise more readily and be perceived incorrectly as gram negative. Some factors, e.g. composition of the growth medium and age of the culture[1] can influence the tendency of gram positive bacteria to decolourise.
Several modifications of the Gram stain procedure have been developed to overcome these decolourisation difficulties.[2] Cerny was able to distinguish gram negative from gram positive facultative bacteria by assaying for amino peptidase, a constitutive enzyme found primarily in gram negative bacteria.[3] Another method for the preliminary classification of bacteria is the use of a 3% solution of potassium hydroxide (KOH). Like the gram stain reaction, the KOH test is based on the differences in the chemistry of the bacterial cell wall. The cell wall of gram negative bacteria is easily disrupted when exposed to dilute alkali solutions.[4] Another rapid method is the testing of susceptibility to vancomycin (5g).[5] In this study, the KOH test is compared with the Gram stain reaction and vancomycin susceptibility test for preliminary classification of gram positive and gram negative bacteria.

 ~ Materials and Methods Top

A total of 218 isolates obtained from various clinical samples were subjected to Gram stain.
KOH String test
A loopful of growth from a colony of the organism was emulsified on the surface of a glass slide in a suspension of 3% KOH. The suspension was stirred continuously for 60 seconds after which the loop was gently pulled from the suspension. The test was considered positive if stringing occurred within the first 30 seconds of mixing the bacteria in KOH solution.
Vancomycin susceptibility test
Using an inoculum corresponding to Kirby Bauer technique[6] a lawn culture was made on Mueller Hinton agar. Vancomycin discs (5g) were placed on the lawn culture and plates were incubated at 37C overnight. Any zone was read as a positive test.
The above tests were also performed using standard gram positive Staphylococcus aureus (NCTC 6571) and gram negative Pseudomonas aeruginosa (NCTC 10662) strains.

 ~ Results Top

Out of the 218 isolates 44 (20.18%) were gram positive and 174 (79.82%) were gram negative bacteria. Gram positive isolates include Staphylococcus spp., Streptococcus spp. and Enterococcus spp. Gram negative rods included Pseudomonas spp., Klebsiella spp., Proteus spp.,  Escherichia More Details coli, Enterobacter spp.,  Salmonella More Details typhi,  Salmonella More Details paratyphi and Acinetobacter spp. [Table - 1].
[Table - 2] indicates the results of the KOH string test and vancomycin test of all the isolates tested. The standard strains Staphylococcus aureus   (NCTC 6571) was vancomycin sensitive and negative for KOH string test, while that of Pseudomonas aeruginosa (NCTC 10662) was vancomycin resistant and positive for KOH test.

 ~ Discussion Top

The preliminary classification of a bacterium as gram positive or gram negative is an essential step in both diagnostic microbiology and clinical medicine. The Gram stain is generally the first procedure performed in the identification of a bacterium and the results of this differential stain often determine the subsequent identification procedures. Often Gram stain yields equivocal results particularly with gram positive anaerobic bacteria and anaerobic gram negative bacteria.[7]
In our study, all isolates of gram positive bacteria were susceptible to vancomycin (>6mm zone diameter) and negative for KOH test (100%). Differences in KOH solubility have been used successfully to categorise aerobic and facultative bacteria including bacteria which display variable gram staining reactions. Of the gram negative bacteria 173 (99.42%) were resistant to vancomycin while 1 (0.58%) Acinetobacter calcoaceticus was sensitive to vancomycin (15 mm). Among the gram negatives KOH string test was positive in 172 isolates (98.85%) while 2 (1.15%) Acinetobacter calcoaceticus were negative for stringing.  Moraxella More Details spp. and Acinetobacter spp. may yield false results in both tests but are easily suspected from the presence of diplobacillary nonmotile forms in liquid and the coccoid forms on solid media.[8]
KOH string test and vancomycin tests are simple, inexpensive tests for rapid identification of bacterial cultures. In laboratories where large number of cultures have to be processed, the above two alternative tests may be used in addition to Gram stain for preliminary differentiation.

 ~ Acknowledgement Top

This work was carried out by K.Arthi as part of the student research fellowship awarded to her by the Indian Council of Medical Research during Aug'2001-Sep'2001 in the Department of Microbiology, PSGIMS, Coimbatore. 

 ~ References Top

1.Conn HJ, Bartholomew JW, Jennison MW. Staining Methods. In: HJ Conn and MW Jennison. Manual of Microbiological methods. (McGraw - Hill Book Co., New York) 1957:10-18.  Back to cited text no. 1    
2.Spengler MG, Rodheaver T, Richter L, Edgerton MT, Edlich RF. The Gram stain - the most important diagnostic test in infection. J Am Coll Emergency Physicians 1978;7:434-438.  Back to cited text no. 2    
3.Cerny G. 1976 Method for distinction of gram negative from gram positive bacteria. Eur J Appl Microbiol 1976;3:223 - 225.  Back to cited text no. 3    
4.Davis BD, Dulbecco R, Eiser HN, Ginsberg HS, Wood WB. Microbiology (Harper & Row, Publishers, New York) 1968:31.  Back to cited text no. 4    
5.Halebian S, Harris B, Finegold SM, Rolfe RD. Rapid method that aids in distinguishing gram positive from gram negative anaerobic bacteria. J Clin Microbiol 1981;13:444 - 448.  Back to cited text no. 5    
6.Sutter VL, Citron DM, Finegold SM. Wadsworth anaerobic bacteriology manual, 3rd ed. (The CV Mosby Co., St .Louis) 1980.  Back to cited text no. 6    
7.Gregerson T. Rapid method for distinction of gram negative from gram positive bacteria. Eur J Appl Microbiol Biotechnol 1978;5:123-127.  Back to cited text no. 7    
8.Von Graevenitz A, Greenwood JR, Price K. Miscellaneous gram negative rods. In: Balows A, Hausler WJ, Jr. Ed. Bacterial, mycotic and parasitic infections, 6th ed. (American Public Health Association, Washington, DC) 1981:397-429.  Back to cited text no. 8    
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2004 - Indian Journal of Medical Microbiology
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