Isolation and Identification of Gram Positive and Negative Bacteria from Dairy Department Waste Lines at Baraton University

Introduction

Barton University dairy farm is a source of delicious and nutri-tious milk, one of its kinds. The milk is consumed as yoghurt, mala, ice cream and fresh milk by the students, residential facul-ty members and to the rest of the Barton Community. As a sys¬tem, the farm intentionally inoculates bacteria for production of yoghurt and mala. While non-intention & by nature providenc¬es, some bacteria grow and carry out the metabolic activities.

The dairy department is composed of three major facilities: the processing, milking and cattle feeding & resting structures. Mainly the cattle feed on hay made of maize stalk from the farm. The drainage from these facilities meets at some point. The three units are sources of enteric bacteria, mycobacterium and other parasites that are associated with cattle breeding [3].

The study primarily is to establish a pure culture of gram pos-itive and negative bacteria and use special staining, assays for specific activities & microbial enzymes, effects of disinfectants and antibiotic micro-organism, selective and differential me¬dia and culture characteristics such as oxygen requirement for organism growth, organism motility & haemolysis pattern to identify the taxonomical name of the bacteria found at the dairy department waste system.

Materials and Methods

The aseptic technique was used in dipping a sterile swab into the specimen from the waste drainage and inoculated into TSB culture media. Three inoculated culture media were established; culture A and B were incubated at 370C while culture C was kept at room temperatures to determine the growth of the organ¬ism at ambient temperature for 48 hours.

Three plates of TSA culture were obtain to established colonies and a gram stain was done to acquire a gram positive and neg¬ative bacteria by heat fixing, application of crystal violet and using iodine solution as mordant, thereafter a decolourization was carried out and finally counter stained with safranin . The cell shape and arrangement were determined through the use of a microscope. Parts of the stained colonies were inoculated by use of quadrant streaking to create pure cultures of gram positive and negative of unknown microorganisms [1].

An acid fast stain was applied to both gram positive and neg¬ative bacteria by use of Kinyoun’s carbolfuchsin, decolorized with Acid – alcohol solution and counter stained with Leoffler’s methylene blue and exposed to steam & heat control. The ap¬plications of Acid fast stain to confirm the presence of myco¬bacterium spp. identified in the cause of Tuberculosis in both cattle and human [2, 5]. To ascertain the formation of endospore in bacteria vegetative cells such as Bacillus anthracis, schaeffer Fulton is the method, which involves fixed slides for two cul-tures, saturated with Malachite Green for 10 minutes by use of steam, rinsed and counter stained with Safranin [4, 6].

The two cultures were tested for acid and gas production from carbohydrate fermentation, mixed acid fermentation pathways, presence of acetone and citrate utilization through the use of lactose broth, Voges-Proskaurer test and Simmons citrate re¬spectively. The production of enzymes: catalase, oxidase, phe¬nylalanine, deaminase, tryptophanase, amylase, and gelatinase were tested. Selective and differential media was used to ascer¬tain whether the unknown organisms are lactose, mannitol, and sucrose & glucose fermenters.

Special features such as oxygen requirement for organisms’ growth, their motility and haemolysis pattern were tested through growth analysis on thioglycollate broth, motility media and Blood Agar Plates. The susceptibility to disinfectant of the two cultures was deter¬mined by exposure to 10% omo, 10% jik, 10% dettol and 70% ethyl alcohol. The Kirby bauer disk method was also carried to determine the susceptibility of ampicillin, lincomycin, penincil¬in, minocycline, erythromycin, chloramphenical and co-trimox-azole.

Results

The conjunction point of the drainage constituted of green and turbid sewage that produced a strong odour. Apart from the cat¬tle waste, a combat liquid soap was evident in the sewage. It was an attractive site to investigate the bacteria growth. Inoculation of the specimen in TSB produced a saturated TBS with bacteria growth. Three TSA plate displayed different colonies in size, co¬lour and shape. Gram-positive presenting large single blue rods, while gram negative produced short single rods. The selections were picked out of the numerous colonies on TSA plates.

                      Table 1: Show Casing Tests Results and Characteristics of Gram Positive and Negative Bacteria

Special staining for Acid fast generated bright shinny rods from gram-positive culture while blue rods from gram negative cul-ture. An Endospore stain, microscopy observation demonstrated free spores and endospores in vegetative cell stained blue green for gram positive and reddish pink cell for gram negative.

Both gram positive and negative bacteria fermented lactose & produced gas and carried out mixed acid fermentation pathways but did not ferment mannitol, do not utilise citrate, nor produce acetoine. Among the enzymes tested, the two organisms do not produce phenylalanine deaminase, amylase and urease but gen¬erate tryptophans.

The susceptibility test demonstrated gram-positive resistance to all detergents, lincomycin, penincilin and minocycline. Inhibi¬tion zones were not observed around disks of the detergents and antibiotics. Inhibition zones were illustrated by some antibiotics A (1cm), Mi (1.8cm),E (1.6cm), CO (2.5cm), and C (2cm). The gram negative exhibited no inhibition zones with ethyl alcohol, L,P, and M. While demonstrated inhibitions zones with omo (1.7cm), jik (1cm), dettol (1.1cm), A(1cm),Mi(2.5), E(1.6cm), CO(1cm), and C(2.6cm).

The organisms are motile, facultative and illustrated gamma haemolysis for gram positive and beta haemolysis for gram neg-ative.

Discussion and Conclusion

The organisms’ habitat detects presence of gram positive and negative enterobacteriaceae group of bacteria, whereby most of these bacteria carry out mixed acid fermentation and grow at low PH detected in Methyl red tests. VP test is used widely to classify enterobacteriaceae strains, those bacteria that produce acetoin are VP test positive. In this case, acetoin increase the PH by reducing the acid in the environment or medium [8, 21]. Some cultures, with prolonged time of growth (to seven days) have tested VP positive. As a result, acidity is reduced in the me-dia [7]. Barry and Feeney declares that acetoin can be detected a little faster if creative was added to MR VP broth followed by Barrett reagents [9].

Endospore formation on gram-positive cells has an ability to create resistance to some antibiotics and detergents as described in the results, there were no inhibition zones. The spores and endospore were quite evident in both Endospore and Acid Fast Staining. Acid Fast Staining of gram-positive bacteria demon¬strated refractive bodies of endospore and free spores [6].

Ampicillin is used as plasmid maintaining agent during Esche-richia coli MG 1655 cultivation [7]. Ampicillin does not neces-sitate limitation of growth but plasmid transfer as demonstrated by lack of inhibition zones in gram positive and negative. Lac¬tic acid bacteria can be used as antibiotic against E. coli and Klebsialla spp. Escherichia coli is the most resistant bacteria and therefore lactic acid bacteria antibiotic activities is a solu¬tion to antibiotic resistant that is transmitted to Human popula¬tion [10]. The susceptibility to antibiotic greatly depends on the environment, therefore to determine the resistance of bacteria on host, requires an environment resembling the host [13]. Pre exposure to some antibiotics or detergents can create resistance of bacteria towards other antibiotics or detergents [15].

Escherichia coli are used as a control for detection of trypto-phanase. According to Newton and Shell tryptophanase in Esch-erichia coli catalyses this reaction L-Tryptophan + H20 - indole + pyruvate + NH3 [10, 14].

In normal circumstances and anatomical structure of Escherich¬ia, coli do not support acetoin production and citrate utilization. Therefore, VP and citrate tests are supposable negative. The transfers of plasmid in Isolates of different species and environ¬ment have proved utilization of citrate. Generation of acetoin by Escherichia coli strains in vegetables and fruits due to acquisi¬tion of buidAB gene alter the normal functionality of Escherich¬ia coli. These two phenomena leads to misdiagnosis of E. coli [7, 17].

Presence of catalase reinforces the facultative ability demon¬strated the thioglycollate broth as most bacteria from the fam¬ily of Enterobacteriaceae that are either aerobic or facultative produce catalase enzymes [11]. Escherichia Coli exhibit a num¬ber of cytochrome oxidase such as cytochrome c oxidase; cyto¬chrome bed oxidase and cytochrome boo oxidase that aid it to survive in different habitat. Cytochrome bed oxidase has proved to be pathogenetic enhancer when exposed to NO and enhances respiration in hydrogen sulphide environment [12, 16]. Trypto-phan utilization by tryptophans generates sulphide in Escherich¬ia coli that triggers cytochrome bed oxidase mechanism [16].

In the experiment, the two bacteria culture indicated movement, their growth extended away from the inoculation line. E coli have capabilities of swimming and swarming through the envi¬ronment by use of flagella and as a colony in search of energy and detecting of some substances [19, 20].

The results presented two types of haemolysis; Gamma hae-molysis for gram-positive bacteria, which indicates the absenc¬es red blood, cells lysing due to lack of toxin production. Beta haemolysis in gram-negative bacteria is due to toxin production. E. coli strains such as Escherichia coli O157 enter pathogenic Escherichia coli and Shiga Toxin-Producing E. coli are sources of toxin that lyses red blood cells [22-24].

Author’s Contribution

Stella carried out the experiments and authored this article.

Conflict of Interest

There is no conflict of interest

Funding

This research received no external funding.

References

1. Forster, S., Snape, J. R., Lappin-Scott, H. M., & Porter, J. (2002). Simultaneous fluorescent gram staining and activity assessment of activated sludge bacteria. Applied and envi­ronmental microbiology, 68(10), 4772-4779.
2. Fèvre, E. M., de Glanville, W. A., Thomas, L. F., Cook, E. A., Kariuki, S., & Wamae, C. N. (2017). An integrated study of human and animal infectious disease in the Lake Victo­ria crescent smallholder crop-livestock production system, Kenya. BMC infectious diseases, 17(1), 1-14.
3. Al-Saqur, I. M., Al-Thwani, A. N., Al-Attar, I. M., & Al-Mashhadani, M. S. (2016). Evaluation the virulence of Mycobacterium bovis isolated from milk samples through histopathological study in laboratory animals. International Journal of Mycobacteriology, 5, S90-S91.
4. Oktari, A., Supriatin, Y., Kamal, M., & Syafrullah, H. (2017, February). The bacterial endospore stain on Schaeffer Ful­ton using variation of methylene blue solution. In Journal of Physics: Conference Series (Vol. 812, No. 1, p. 012066). IOP Publishing.
5. Nalapa, D. P., Muwonge, A., Kankya, C., & Olea-Popel-ka, F. (2017). Prevalence of tuberculous lesion in cattle slaughtered in Mubende district, Uganda. BMC veterinaryresearch, 13(1), 1-8.
6. Hussey, M. A., & Zayaitz, A. (2007). Endospore stain pro­tocol. Am Soc Microbiol, 8, 1-11.
7. Vivijs, B., Moons, P., Aertsen, A., & Michiels, C. W. (2014). Acetoin synthesis acquisition favors Escherichia coli growth at low pH. Applied and environmental microbiolo­gy, 80(19), 6054-6061.
8. Klaus Bryn., Jan C., Ulstrup., and Fredrik C., Størmer. (1973). Effect of Acetate upon the Formation of Acetoin in Klebsiella and Enterobacter and its Possible Practical Ap­plication in a Rapid Voges-Proskauer Test. Appld Microbi­ology. 25(3):511-512.
9. Barry, A. L., & Feeney, K. L. (1967). Two quick methods for Voges-Proskauer test. Applied microbiology, 15(5), 1138-1141.
10. Adeniyi, B. A., Adetoye, A., & Ayeni, F. A. (2015). Anti­bacterial activities of lactic acid bacteria isolated from cow faeces against potential enteric pathogens. African health sciences, 15(3), 888-895.
11. Taylor, W. I., & Achanzar, D. (1972). Catalase test as an aid to the identification of Enterobacteriaceae. Applied micro­biology, 24(1), 58-61.
12. Giuffrè, A., Borisov, V. B., Mastronicola, D., Sarti, P., & Forte, E. (2012). Cytochrome bd oxidase and nitric oxide: from reaction mechanisms to bacterial physiology. FEBS letters, 586(5), 622-629.
13. Zachary D., Silpak B., Raghavendra G., and David G. R. (2016).Antimicrobial Susceptibility of Enteric Gram Nega­tive Facultative Anaerobe Bacilli in Aerobic versus Anaero­bic Condition. Plos One. 11(5): 1-12.
14. Newton, W. A., & Snell, E. E. (1964). Catalytic properties of tryptophanase, a multifunctional pyridoxal phosphate en­zyme. Proceedings of the National Academy of Sciences, 51(3), 382-389.
15. Liu, G., Ding, L., Han, B., Piepers, S., Naqvi, S. A., Barke-ma, H. W., & Gao, J. (2018). Characteristics of Escherich­ia coli isolated from bovine mastitis exposed to submini-mum inhibitory concentrations of cefalotin or ceftazidime. BioMed Research International, 2018.
16. Korshunov, S., Imlay, K. R., & Imlay, J. A. (2016). The cytochrome bd oxidase of Escherichia coli prevents respi­ratory inhibition by endogenous and exogenous hydrogen sulfide. Molecular microbiology, 101(1), 62-77.
17. Ishiguro, N., & Sato, G. (1979). The distribution of plas­mids determining citrate utilization in citrate-positive vari­ants of Escherichia coli from humans, domestic animals, feral birds and environments. Epidemiology & Infection, 83(2), 331-344.
18. Fay, G. D., & Barry, A. L. (1974). Methods for detecting indole production by gram-negative nonsporeforming an-aerobes. Applied Microbiology, 27(3), 562-565.
19. Ravichandar, J. D., Bower, A. G., Julius, A. A., & Collins,C. H. (2017). Transcriptional control of motility enables di­rectional movement of Escherichia coli in a signal gradient. Scientific reports, 7(1), 1-14.
20. Gómez-Gómez, J. M., Manfredi, C., Alonso, J. C., & Blázquez, J. (2007). A novel role for RecA under non-stress: promotion of swarming motility in Escherichia coli K-12. BMC biology, 5(1), 1-15.
21. Qadri, S. M., Nichols, C. W., Qadri, S. G., & Villarreal, A. (1978). Rapid test for acetyl-methyl-carbinol formation by Enterobacteriaceae. Journal of Clinical Microbiology, 8(4), 463-464.
22. Buxton, R. (2005). Blood agar plates and hemolysis proto­cols. American Society for Microbiology, 1-9.
23. Hornitzky, M. A., Mercieca, K., Bettelheim, K. A., & Djordjevic, S. P. (2005). Bovine feces from animals with gastrointestinal infections are a source of serologically di­verse atypical enteropathogenic Escherichia coli and Shi­ga toxin-producing E. coli strains that commonly possess intimin. Applied and environmental microbiology, 71(7), 3405-3412.
24. Sata, S., Fujisawa, T., Osawa, R., Iguchi, A., Yamai, S., & Shimada, T. (2003). An improved enrichment broth for iso­lation of Escherichia coli O157, with specific reference to starved cells, from radish sprouts. Applied and environmen­tal microbiology, 69(3), 1858-1860.