This work of identification of bacterium was performed through different biochemical tests. Unknown cultures were streaked on EMB, SS and MacConkey agar plates to distinguish the lactose and lactose non-0feremnetros and to identify the grams nature of the culture.
The culture was also streaked on TSI agar slants to confirm the cultures ability to switch n from glucose to lactose and sucrose.Performing the biochemical tests for the given bacterium it is confirmed that the cultures provided were Gram-negative E.Coli, Gram-negative paracoliform Yersinia enterocolitica and Gram positive Bacilli. Introduction:Microorganisms are omnipresent, and plays crucial role in biogeochemical cycles.
In human perspective they are classified as pathogenic (disease causing) and non pathogenic organism (Pommerville). Once pathogenic organism succeeds to colonize in side the body it starts exerting its effect by interfering normal metabolic processes. In most of the infection there are specific symptoms associated with it but in certain cases it is difficult to distinguish two types of infection causes by two different organisms and in this case detail microbiological investigation is needed to identify causative agent and than its cure (Pelczar).
There are different ways by which one can identifies particular organism like based on its morphology, Staining behavior, air requirement for respiration, substrate utilization etc. , termed as biochemical test. Here we have performed various biochemical tests for identification of given gram positive and gram negative organisms.
Materials and methods
- The given cultures were streakek on. TSA, Blood agar and Maconkey agar plates and incubated at 37° C.
- After incubation plates were observed for growth and results were noted down.
- Single colony were picked up and used to prepare smear for gram staining.
- Based on results of gram staining cultures were classified as Gram positive and Gram Negative.
- Based on results of initial streaking and gram’s staining cultures were inoculated in different selective or differential media for further identification.
- Hemolytic, gram’s positive cocci was inoculated to differential media namely mannitol salt agar to distingsh between streptococci and staphylococci.
- Similarly for catalase assay, single colony from positive blood agar plate was used to make smear on glass slide. The smear was overlaid by H2O2 and observed for bubble formation.
- Isolated colony was picked up from MacConkey agar plate and streak on to the EMB ,TSI slant and SS slants ( streaking and stub culturing)
- Similarly, isolated colonies from all three plates were streaked on galantine, starch, casein and citrate containing plates to determine gelatinase, amylase, proteases and citrate utilization capability of organisms.
- All the plates and slants were incubated at 37°C.11) After incubation plates and slants were taken out and results were noted forpositive and negative results.
- Based on standard result chart unknown organisms ware identified.
- Culture 1: The culture displayed negative for caseinase, amylase and gelatinase. On EMB medium colonies appeared blue-black with metallic green sheen. The colony formation was mostly inhibited on SS medium. On MacConkey agar plate bright pink colonies of the culture was seen. TSI slant displayed interesting result; both the slant and the butt were found to be yellow whereas no hydrogen sulphide production was observed. The citrate medium displayed green color. The blood agar and the catalase test were not performed. No growth of this culture was seen in the citrate slants. No growth was seen on MSA medium.
- Culture 2: The caseinase, amylase and gelatinase assay were not performed for this culture. The EMB medium displayed no colour. Colorless colonies were seen in SS medium. The stub of the TSI slant appeared pink in colour, but the butt was yellow in colour. No gas bubbles were observed and H2S production was also negative. No growth was seen on the citrate slants. The blood agar and the catalase test were not performed. No growth was seen on MSA medium.
- Culture 3: The gelatinase assays were not performed for this culture. Clear zones around the vicinity of the growth was seen for both caseinase and amylase test. The growth was inhibited in EMB, SS and MacConkey agar plate. The blood, MSA and catalase tests were not performed.
Culture 1: The culture was unable to produce caseinase, amylase and gelatinase enzyme. Colored colonies especially blue-black on EMB medium is a sure indication that this culture is able to utilize lactose as the sole source of carbon and the lactose and the dye were not inhibitory thus clearly indicating that this culture is Gram-negative. Moreover, this culture appeared blue-black with metallic sheen further indicates that the culture could be E.Coli, a gram-negative bacterium. No growth on SS medium also indicates that this culture is not salmonella or shigella species.
Colonies in Macconkey agar plate is a sure sign that that this culture is Gram-negative and possess enzymes to breakdown lactose into glucose and galactose. Moreover, the pink colonies are clear indication that this bacterium is an enteric coliform. Both the slant and the butt appeared yellow on the TSI slant display the acidic nature of the medium. This culture is able to utilize glucose, since it is present in lower concentration, after its deprivation it also switches over to lactose and sucrose, thus finding other means of respiration.
The acidic nature (yellow) of both slant and the butt is because of the utilization of the sugar. The bacterium is not able to utilize citrate as its sole source of carbon. All the above results indicate that this culture is a Gram-negative bacterium essentially an enteric coliform. The blue black growth with metallic sheen on EMB medium and pink colonies on Macconkey agar respectively further confirms that the culture is Escherichia coli.
Culture 2: Colonies on EMB medium indicates that this culture could be Gram-negative; since it cannot utilize lactose, thus appears colorless. Colorless colonies on SS media also indicate that this culture is not shigella or salmonella but, also is lactose non-fermentor. Furthermore, colorless colonies on Macconkey agar conforms that this culture is gram-positive in nature and is a paracolon. The pink TSI slant and the yellow butt indicate the alkaline/acidic nature of the TSI slant.
Alkaline nature clearly indicates that only glucose was utilized/fermented by this culture and this culture is essentially a lactose non-fermentor. Since the culture cannot utilize other carbon source other than glucose, it further utilized the proteins and amino acids thus turning the slant alkaline. The culture could not utilize citrate as the sole carbon source. All the above indicates and results on Macconkey and EB further conforms that the culture is essentially gram-positive paracolon, Yersinia enterocolitica.
Culture 3: This culture is essentially a Gram-positive Bacilli since it is also caseinase and amylase positive. Moreover, it displays no growth on EMB, SS and MacConkey agar, which further indicates that it is not an enetrococci and since it hemolysin negative, it is also not form the staphylococcal group of organism Conclusions:The unknown cultures were identified as Gram negative E.coli, Gram negative Yersinia enterocolitica and gram positive bacilli.
- Pelczar, Reid & Chan. Microbiology. Tata-macgrow Hill, 1996.
- Pommerville, Jeffrey C. Alcamo’s Fundamentals of Microbiology. Jones and Bartlett Publishers, 2008.