Identification Research of Serratia Marcescens

This test uses the process of hydrolysis of casein as an indispensable aid in the identification of Serratia marcescens. The experiment examined a total of 300 strains from the household of Pseudomonadaceae and Enterobacteriaceae. The organism in question could decompose casein whilst the other bacteria failed to reason any hydrolysis and only went to the extent of producing hazy zones. Previous research and the test indicate that the casein hydrolysis experiment is beneficial in the identification analysis of the gram-negative bacteria Serratia marcescens.

The isolation of the gram-negative microorganism in the genera Proteus, Serratia, Aeromonas and Pseudomonas in a laboratory is not an easy task. There are many experiments used for the separation and determination of the bacterial group mentioned above. The test only adds a few observations which have proved useful in the successful identification of the non-pigmented strains of Serratia marcescens (Graevenitz & Buchholz, 1971). The organism has shown an easily notable and regenerating ability to hydrolyze casein in milk agar. The reaction is significantly different from that of Pseudomonas aeruginosa.

The experiment has demonstrated the capability of three bacterial strains to cause hydrolysis of milk components. Serratia marcescens had a 97% capability, Pseudomonas aeruginosa 99% while Proteus mirabilis was 94%.

Introduction

Serratia marcescens is a gram-negative bacillus in the family Enterobacteriaceae and the primary cause of hospital-related infections (Grimont & Grimont, 1984). The organism under study has had various names such as Chromobacterium prodigiosum but Bizio coined the current term Serratia marcescens in 1823.

There have been many studies relating to isolation, identification, and pathogenicity of Serratia marcescens. Precise identification of this organism is vital in determining the causative factors of disease outbreaks. Existing investigations were carried out about water and specific plastics such as blood bags (Hejazi & Falkiner, 1997). The API 20E system has been the method for identification of the bacteria. However, over time, the technique was discovered to identify only 85% of the gram-negative bacillus correctly. For the API 20E method to be accurate, there have to be other sugar tests such as arabinose and raffinose.

Other tests used in the identification of this organism include the test of methyl red that evaluates whether a bacterium undergoes fermentation in mixed-acids; Serratia marcescens produces a negative result. Serratia marcescens test is also negative for lactic acid due to oxidative and fermentative metabolism. For nitrates, the outcome is positive. The red pigmentation of Serratia marcescens is a vital distinguishing factor when making a comparison with some strains.

Materials and methods

Casein hydrolysis is commonly used to analyze whether an organism can produce caseinase, an enzyme which disintegrates casein usually found in milk to soluble products. The casein has numerous protein components which forms a part of amino acids containing 85% of milk proteins. It is also responsible for the white color of milk. For utilization of casein, bacteria cells produce proteolytic exoenzymes which are a combination of caseinases and peptidases (Cappuccino & Sherman, 2008). There is secretion of exoenzymes outside the cells that then hydrolyze proteins to amino acids

The hydrolysis of casein entails the culturing of a bacterium on skimmed milk to produce soluble amino acids. The plates are then regularly checked for hydrolysis. In dishes where hydrolysis has not taken place, the result is white while the plates where hydrolysis has occurred there are clearing zones around the growth (Zimbro et al., 2009). The purpose of this test is to determine the breakdown of milk proteins by proteolytic exoenzymes produced outside the cell by the various bacteria strains under study.

The use of milk is a relatively affordable option for any laboratory seeking to undertake this test of bacteriology screening. The preparation of casein medium (Ajello et al., 1963) is as follows:

Skim milk 10g and distilled water 100ml

Distilled water 100ml

agar 2g and distilled water 100ml

There was heating of both solutions at 1200 C then cooled separately to around 450C. After cooling, both mixtures were poured into a Petri dish. All organisms under test were introduced in the skim milk agar by point inoculation (Salisbury & Likos. 1972). Therefore, a heated loop was used to inoculate one side of the plate with a 2.5cm line; 6-8 isolates may be cultured on one dish. The cultures were then put under incubation upside down for 24 hours at temperatures of about 35 to 37o C. There was an examination of specimens for traces of casein hydrolysis. The Petri dishes underwent further incubation at 370C for another 24 hours.

The results were as follows

Organism

No. tested

24-hr reading at 35-370C

48-hr reading at room temperature

Result

Serratia marcescens

52

5(RH)

12(RH)

three failed to produce a clearing

Pseudomonas aeruginosa

80

6(RH)

12(RH)

one did not produce a clearing

Proteus vulgaris

8

3(SH)

6(RH)

Produced hazy zones

Esch. Coli

12

0

0

Produced hazy zones only

Salmonella group A

5

0

0

Only hazy zones

Ent hafnia

7

0

2

Hazy zones only

Proteus mirabilis

40

3(SH)

6

Two produced a clearing

Ent. Aeroganes

25

0

0

Only hazy zones

Herellea vaginicola

25

0

0

Proteus morganii

10

0

0

Proteus vettgeri

11

0

0

Alk. Foecalis

15

0

0

One produced a clearing

Klebsiella pnumoniae

50

0

0

SH-Slow hydrolysis RH-Rapid hydrolysis

The cultures without the final result did not produce any clearing or hazy zones. Some bacteria strains did not undergo incubation for 48 hours because of the spread and fast undergrowth of various isolates.

Discussion

The table demonstrates a comparison of the capability of various bacterial strains from the Enterobacteriaceae and Pseudomonadaceae families to hydrolyze casein in 24-48 hours. From the results, Pseudomonas aeruginosa and Serratia marcescens produced clear transparent zones while others produced hazy zones or no hydrolysis at all. Serratia marcescens used extracellular enzymes to disintegrate the peptide bonds in casein. As indicated in the table, the hydrolysis occurred rapidly. There were a few aberrant outcomes from the experiment.

From this experiment, it is evident that hydrolysis of casein is essential in differentiating and identifying Serratia marcescens.

References

Ajello, L., Georg, L.K., Kaplan. W., and Kaufman, L. (1963). Laboratory manual for medical Mycology (Public Health Service Application, no. 994). Communicable Diseases Center, Atlanta, Georgia.

Cappuccino J.G. & Sherman N. (2008). Microbiology: A Laboratory Manual, 8th ed. Pearson Benjamin Cummings, San Francisco, CA, USA

Graevenitz Von & Buchholz D, (1971) Changes in their causative agents Trends and possible basis, Springer

Grimont PAD & Grimont F.Genus (1984)VIII. Serratia. In: Krieg NR, Holt JG (eds) Bergey’s manual of systematic bacteriology, vol l.Baltimore, Williams and Wilkins.:477-484

Hejazi, A., & Falkiner, F. (1997). Serratia marcescens. Microbiologyresearch.org. Retrieved 20 November 2017, from http://www.microbiologyresearch.org/docserver/fulltext/jmm/46/11/medmicro-46-11-903.pdf?expires=1511185744&id=id&accname=guest&checksum=F890AB2BA049DA05C6FCD0000387201C

Salisbury, W., & Likos, J. (1972). Hydrolysis of casein: a differential aid for the identification of Serratia marcescens. jOURNAL OF CLINICAL PATHOLOGY. Retrieved 20 November 2017, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC477626

Zimbro M.J, Power D.A., Miller S.M., Wilson G.E, & Johnson J.A (2009). Difco &BBL Manual Manual of Microbiological Culture Media, 2nd ed.