Screening and Identification of Bacteria from Vellore Polycarbonate Lenses

 

Roshan Hari, Tanmay Talwalkar, Suneetha V

School of Biosciences and Technology, VIT University, Vellore-632014

 

ABSTRACT:

Spectacles are used by millions of people every day. Although it is an item of daily use for many, not all feel the need to clean their glasses daily^[1]. As, most of today’s lenses in use are made of polycarbonate, this experiment aims at studying the various organisms present on polycarbonate lenses. From staining, Biochemical assay tests and other analytical techniques, different species of bacteria are noted and finally identified. Shigella spp. were identified as the major microbial contanimant on these polycarbonate lenses. Shigella most commonly causes shigellosis and dysentry in humans^[2].

 

 

 

INTRODUCTION:

Polycarbonate is a thermoplastic polymer. Due to it’s strength and lightweight frame It finds many uses. One of it’s many uses comes in the spectacle lens industry. These are mainly used in rimless frames.

 

Our focus of this study is to screen microbes and identity. Hence this type of research is rudimentary^[2][3][4].

l      Rod shaped

l      Gram negative

l      Non-spore forming

l      Non motile

l      Facultative anaerobe.

 

It primarily causes dysentery in humans and one of the most widely distributed disease causing bacterial genus. It is closely related to the genus of E.coli.

 

 

 

 

 

MATERIALS AND METHODS:

Sample collection^[3]:

Samples were collected form three scholars at the Vellore Institute of technology in Vellore. They are residents of the hostel premises in Vellore. The samples are collected from the spectacle lenses in daily use by them. The samples were collected at ambient temperatures of 29-31^oC. By using clean synthetic gloves and sterile swab sample is collected from spectacle lenses in a laminar air flow chamber. After collection samples were taken for lab processing.

 

Methodology of Process:

Selection of sample donors

Nutrient Agar is prepared and sterilised

Required glasswares is sterilised

Agar is poured on petri plates and allowed to cool

The sample is streaked on the Agar containing Petri plates.

 

Media:

The following preocedure was followed for preparing nutrient Agar medium.

 

(a) Nutrient Agar:

Nutrient agar was prepared and sterlised.

2.8 grams of nutrient agar was used along with 2.0 grams of agar agar. Per 100 ml of prepared media.

Colour obtained is pale yellow. The pH of the medium was 7.2.

 

Streaking:

After swabbing the sample lenses with a sterile swab it was streaked on the prepared agar media. After streaking the petri plates were incubated in an incubator at 37°C for 48 hours.

 

Note:

It is note worthy that the samples that were streaked in the media show bacterial colonies growth.

 

Morphology Study By staining:

Simple staining^[4]:

The samples collected are stained using methylene blue as dye. They were observed under 40x. The observations are pictographically depicted below.

 

Gram staining^[5]:

The culture that was obtained was stained using Gram’s staining technique. Methylene blue was used as the primary stain and safranin was used as the counter stain. The observations are pictographically depicted below.

 

PROCEDURE in brief:

Smear was made using an inoculation loop

 

Mehtylene blue was added

Iodine was used as mordant

It is now washed with decolouriser(Acetone)

It was counterstained with safranin

Observations are noted and pictures were taken

 

Biochemical assay and Sugar fermentation test (IMVIC Test) :

The following tests were carried out in a prepared assay kit.^[2]

 

1.Biochemical Indole test^[8]

2.Biochemical Methyl Red Test^[9]

3.Biochemical Voges-Proskauer Test^[10]

4.Biochemical Citrate utilization^[11]

5.Sugar [Glucose] fermenatation

6.Sugar [Arabinose] fermenatation

7.Sugar [Lactose] fermentation

8.Sugar [Sorbitol] fermentation

9.Sugar [Mannitol] fermentation

10.Sugar [Rhamnose] fermentation

11.Sugar [Sucrose] fermentation

 

Catalase test^[6][7]:

Hydrogen peroxide was kept on a slide, the culture was introduced and slightly agitated. If there is presence of catalase foaming will be observed and in the absence of catalase no characteristic changes are observed.^[6]

 

Table 1.Observation made in the IMViC test

Test		Results
Indole Test		-
Voges-Proskauer Test		-
Fermentation of sugar test	Glucose	-
	Sucrose	-
	Lactose	-
Mannitol test		-
Methyl red test		+
Arabinose Test		-
Sorbitol Test		-
Rhamnose Test		-

 

RESULT AND DISCUSSION:

Isolation of bacteria:

Bacteria were isolated from three sets of polycarbonate lenses. They were swabbed onto a nutrient Agar plate.After incubation for 48 hours a white growth was obtained on the nutrient agar.

 

Staining of Bacteria:

1.Simple staining:

Simple staining is done to observe the external shape and morphology of a bacterium. This was done with methylene blue as a primary stain^[4].The following was observed under 40x objective of the microscope

For sample A: several rod shaped batceria observed along with a few round shaped bacteria in groups.

For sample B :several rod shaped bateria observed along with a few spiral shaped bacteria.

 

For sample C:several rod shaped bateria observed along with a few spiral shaped bacteria.

This observation may suggest that the bacteria may be bacillus or Escherichia spp.

 

2.Gram staining:

Gram staining is a method of differentiating bacteria based on their cell wall composition. They are differentiated broadly into two types, Gram positive and gram negative^[5]. Gram positive cells are observed as blue in colour after the staining. Gram negative bacterium appear red. Gram positive bacterial cell wall is composed of peptidoglycan primarily, whereas gram negative bacterial cell wall is composed of peptidoglycan and mucopolysaccharide^[5].The following was observed under 40x objective lens.

 

Sample A: Many rod shaped gram negative cells were observed along with very few round shaped gram negative cells.

Sample B:Large number of gram negative rod shaped cells were observed along with a few gram positive round shaped cells

SampleC: Large number of gram negative rod shaped cells were observed along with a few gram positive round shaped cells.

 

Fig 1.The sample polycarbonate lenses used.

 

 

Fig 2.All three samples.

 

 

Fig 3.Autoclaved glassware

 

 

Fig 4.Prepared Agar media.

 

 

                                        A                                                                             B                                                                                                 C

5,6,7.simple staining :-

1:-Sample A, 2:-Sample B, 3:-Sample C

 

 

Sample A

Fig 8.Differential staining:

 

 

Sample B

Fig 9.Differential staining :

 

 

Sample C

Fig 10.Differential staining

 

 

Fig11.Prepared slides for gram staining

 

 

Fig 12.Observations of the IMViC test

 

 

 

Fig.13 Catalase test observations.

 

3. Biochemical Assay test:

Biochemical assay test is a conformatory test for genus identification.It differentiates based on the primary metabolties and sugar fermentation by the bacteria^[2]. There are 7 different sugars:- Glucose, Arabinose, Lactose, Sorbitol, Mannitol, Rhamnose, Sucrose. These help in identifying the genus.The samples showed positive test result only in the methy red test.

 

4. Catalase test:

No foaming was observed therefore catalase is absent.This eliminates the possibility of presence of Streptococcus species.

 

CONCLUSION:

Based on general shape of the bacteria in the simple staining, exhibtion of gram negative nature, catalase test and based on the results of the Biochemical assay(IMVIC test) we are let to the possible conclusion of that, the main bacterial contaminant on polycarbonate spectate lenses is Shigella spp. It may result in increase in occurrence of dysentery among spectacle users.

 

ACKNOWLEDGEMENTS:

We sincerely express our deepest gratitude to Dr G. Viswanathan, hon’ble Chancellor and founder, VIT (Deemed university), Vellore. We want to express special thanks to  Mr Sankar Viswanathan and Mr G.V. Selvamfor continually motivating us towards success.

 

 

REFERENCES:

1.       Khorazo D, Thompson R. The bacterial flora of the normal conjunctiva. Am J Ophthalmol.  1935;18:1114–1116.

2.       Ananthinarayan, Panicker. Textbook of Microbiology

3.       Prescott’s Microbiology. C.J. Woolverton, J Willey, L. Sherwood.

4.       Microbiology: Principles and Explorations. J.G. Black.

5.       John G. Holt; Noel R. Krieg; Peter H.A. Sneath; James T. Staley; Stanley T. Williams (1994). Bergey's Manual of Determinative Bacteriology (9th ed.). Lippincott Williams and Wilkins. p. 11

6.       Rollins DM (2000-08-01). "Bacterial Pathogen List". BSCI 424 Pathogenic Microbiology. University of Maryland. Retrieved 2009-03-01.

7.       Srinivasa Rao PS, Yamada Y, Leung KY (September 2003). "A major catalase (KatB) that is required for resistance to H2O2 and phagocyte-mediated killing in Edwardsiella tarda". Microbiology. 149 (Pt 9): 2635–44.

8.       Bachoon, Dave S., and Wendy A. Dustman. Microbiology Laboratory Manual. Ed. Michael Stranz. Mason, OH: Cengage Learning, 2008. Exercise 15, "Normal Flora of the Intestinal Tract" Print.

9.       H. T. Clarke; W. R. Kirner (1941). "Methyl Red". Organic Syntheses.; Collective Volume, 1, p. 374

10.     MacFaddin, J. F. 1980. Biochemical Tests for Identification of Medical Bacteria, 2nd ed. Williams and Wilkins, Baltimore

11.     A. Forbes, Betty; Daniel F. Sahm; Alice S.Weissfeld. BAILEY and SCOTT'S Diagnostic Microbiology (tenth ed.). Don Ladig. p. 430.

 

 

 

 

 

Accepted on 18.06.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(9): 3927-3931.