Identification Of Unknown Bacterial Sample: Methods And Results

Aim of the Study

Discuss about the Identification Of An Unknown Sample Of Bacteria.

Background and Significance of the Study

One of the important aspects of the microbiology is identification of the unknown bacterial sample. One of the basic and the sole pillar of the bacterial identification, which have been used in microbiology since time immemorial is Gram’s staining. It classifies bacteria into two groups, gram positive and gram negative based on their cell wall composition. However, with the advancement of technology and research, several different methods and procedures have been elucidated into order to classify the bacteria on the basis of their shape, presence of flagella, endospore, fermenting capability and also on the basis of oxygen requirement or respiration (aerobic and aneorobic bacteria).  

The aim of the study is to elucidate identify unknown bacterial sample based on the application of the several microbiogical identification techniques.

The aim objective of the study is to identify the gram positive/negative strain of the bacteria, Identify the external outline of the bacteria, Identify the lactose fermenting capacity of the bacteria, Identify the peroxidase activity of the bacteria, Identify whether the bacteria is facultative anaerobe, anaerobe, obligate anaerobe or aerobic bacteria.  Differential staining and the use of differential media help to identify specific group or bacteria amidst different strains of bacteria. A bacterium can react differently with nutrient s provided in the differential media and thus helps in the identification. Differential techniques to identify bacteria had been used, like Eosin methylene blue plates, Mannitol agar tests, catalase oxidation and the oxidase tests, depending upon the ability of the unknown bacteria to perform the catalase activity.  The experiment further focuses on the oxidative fermentation of the facultative anaerobe that helps to identify the bacteria. The fermentative property of the bacteria has a wide range of application in the fermentative industry. They have a wide range of application in the food, beverage, and bakery and breweries industry.  The process of fermentation is used in the food processing industry for converting carbohydrates into alcohol. Microorganisms such as yeast or bacteria that employ anaerobic respiration are used in this process. Widely consumed fermented foods that are processed by bacteria are vinegar, cheese, yoghurt. Escherichia coli are used for the production of ethanol, lactate, succinate and acetate.

In this method the bacteria has been subjected to differential staining. Differential staining is normally done to identify a specific strain of bacteria in comparison to the other bacteria.  Different types of bacteria can have different types of reactions with the particular type of stains used. Few techniques that have been used in the identification of the unknown bacteria have been provided.

Objectives

Eosin methylene blue plates- Eosin Methylene blue is used to determine the gram negative enteric rod shaped bacteria hence gram positive and gram negative bacteria can be distinguished from the following differential staining.  Eosin and methylene blue helps in inhibiting the growth of the gram positive bacteria and helps to identify the gram negatives (Wills et al.2014). The two dyes present in this stain helps to indicate the lactose fermentors and the non lactose fermenters. The lactose fermentors produce colonies with clear borders and dark centers. The non lactose fermentors form colorless colonies.

Mannitol agar- The mannitol agar can be used to identify the staphylococci group of bacteria. Staphyloccoccus bacteria cannot use mannitol as their nuytrient and hence produce colorless colonies (Hemraj et al.2013). The group of bacteria that can utilize mannitol can turn the mannitol agar plate yellow and hence can be identified.

Catalase oxidation- Some bacteria produces flavoprotiens that can bring about reduction of oxygen with the production of hydrogen peroxide and superoxide. These free radicals are harmful and they can destroy the bacterial cells (Hemraj et al.2013). There are certain obligate and facultative anaerobes that contain the enzyme superoxide dismutase that helps in the destruction of the super oxide and they can also produce catalase or peroxidase that neutralizes the hydrogen peroxide (Borisov et al.2013)

This catalase activity can be detected by the addition of the hydrogen peroxide a substrate to tryptic soy agar slant. A bacteria producing catalase will be able to produce oxygen case which can be determined by the bubbles released. This is recognized as a useful method in detecting the catalytic activity of bacteria.

2O^2- + 2H+ → O₂ + H₂O₂   (Action of Superoxide dismutase)

2H₂O₂ → 2H₂O + O₂               (Action of Peroxidase)

Oxidase- The oxidase cytochrome test involves addition of an oxygen test strip containing tetramethyl-p-phenylenediaminedihydrocholoride or an Oxidase Disk, p-amino-dimethylaniline. The oxidase test reagent is light pink in color and acts as the substrate and donates electrons to the cytochrome oxidase produced by the bacteria, which can turn the strip to dark purple due to the presence of free oxygen. A dark purple coloration is the indication of positive result.

Oxidation fermentation Test- This test was done to differentiate between the gram positive and the gram-negative bacteria on the basis glucose fermentation (Hemraj et al. 2013).

Further concentration of the bacteria has been measure by diluting them and measuring the optical density at 660 nm.

Methods

The nutrient agar plate over which the unknown bacterial sample was grown overnight at 37 degree centigrade in the incubator showed that the bacteria colonies are circular in shape with a convex elevation and a smooth outer margin.

Gram staining observation of the unknown sample

 Pink coloured colonies had been found which determines gram-negative bacteria.

Metabolic profiling: In the Eosin methylene blue agar, the unknown sample showed growth with a greenish metallic sheen, which indicates Escherichia coli bacteria, which has been able to ferment lactose.

In the Mannitol agar plate, the unknown inoculums showed no change in color.

Catalase activity: Bubbles were formed on the plates as soon as it is reacted with the hydrogen peroxide indicating catalase activity of the bacteria.

Oxidase activity:

The oxidase test reagent showed a dark purple color which indicated a positive test for the bacteria having the oxidase enzyme.

Oxidation/Fermentation test:

Table 1

If the open assay produces yellow coloration then it gives indication that the organism has oxidized as well as fermented. A green coloration signifies no reaction with the glucose. For closed assay, yellow coloration indicates fermentation and a green colour signifies that the bacteria has not reacted with the glucose and has not oxidized. Yellow result is obtained in the unknown sample both in the open and in the closed assay which is same as that of Escherichia coli which indicates that the bacteria is Escherichia coli.

No Oil (open assay)	Oil (closed assay)	Result
yellow	green	Oxidation of glucose
yellow	yellow	Fermentation of glucose
green	green	no action on glucose

Table 2

It can be seen that the colonies obtained from the unknown sample is same as that of the Escherichia coli. The orange colored colonies are due to the production of gluconic acid. The orange colour at the top indicates that the fermentation has occurred at the top due to oxidation and no glucose utilization had taken place at the bottom.

Name of the bacteria	With paraffin	Without Paraffin
Bifidobacterium	Orange colour, colonies visible	All green colored colonies
Pseudomonas fluorescens	Green colonies, no colour visible	Orange top , green bottom
Escherichia coli	Green on bottom, orange on top colonies	Orange on top, green at the bottom,
Unknown bacteria	Green on bottom, orange on top, Escherichia coli colonies visible	Orange on top and green at the bottom , colonies visible

Table 3

The spectrometric results obtained are as follows-

It can be seen as the dilution increases the absorbance at 600 nm decreases as the concentration of bacteria decreases.

Dilution	Absorbance(600 nm)
½	0.224
¼	0.141
1/8	0.100
1/16	0.075

The unknown bacterial sample showed round colonies with convex elevation and a continuous margin. This signifies that the margin of the unknown bacterial sample is smooth and continuous (Harrigan, Wilkie and Margaret 2014). The gram staining of the isolated single concave bacterial colony provided pink color, indicating gram-negative strain of bacteria (Carter et al 2012). The stained slide when viewed under the light microscope, it showed a tubular structure and no motility was detected. The gram-negative rod was then subjected to metabolic profiling in order to detect the lactose fermentation. When plated over Eosin Methylene Blue (EMB) Agar, the gram-negative bacterial sample provided metallic green to blue colour colonies (positive result). The blue color signified that the gram-negative bacterial sample is successful in fermenting lactose. This lactose fermenting gram negative rod was then plated over the mannitol agar and no colour change was observed. This test acts as a second confirmatory test, proving that is a gram negative rod and not a gram negative Staphylococcus coccus (Bautista-Trujillo et al. 2013). The lactose fermenting gram negative rod is then checked for the catalaze activity and it showed positive results with the emission of bubbles, indicating the bacterial sample has active catalaze or peroxidase enzyme enzyme and hence do not fall into the category of the strict anerobes (Iwase et al. 2013). The oxidation fermentation test proved that the bacterium is facultative anaerobe. The unknown bacterial sample reacted in the same way as that of the Escherichia coli in the both the open (no paraffin oil attached at the top) and at the closed (paraffin oil taached) systems. This indicates that the unknown bacterial sample could be Escherichia coli.

Confirmatory tests

The importance of the findings showed that the bacteria is gram negative in nature and that means it has thin layer of peptidoglycan with an outer layer of thick Lipo-polysaccharide membrane (LPS). In EMB agar, the Eosin Y and methylene blue are pH indicator it distinguish between lactose fermenting and nonlactose fermenting bacteria. The lactose fermenters produce dark colonies with clear borders while the non-lactose fermenters produce colourless colonies. 

The importance of Mannitol salt agar test lies in the fact that it hsowed that the sample is not Staphylococci. Mannitol-using bacteria produce yellow colour and the bacteria which do not use Mannitol shows no change in the colony (De Visscher et al. 2014).

The importance of catalse test is, the bacteria, which contains flavoproteins reduce oxygen to produce hydrogen peroxide (H2O2) or superoxide (O2-). H2O2 is an extremely toxic oxidising agent that destroys the cellular constituents. Obligate aerobes/facultative anaerobes contains enzymes, superoxide dismutase that catalyses H2O2 into harmless water and oxygen. 

2O2- + 2H+ → O2 + H2O2 (enzyme: Superoxide dismutase) 

2H2O2 → 2H2O + O2 (enzyme: Catalase or peroxidise) 

Such catalase production and activity can be detected by using substrate for H2O2 or the oxygen thus produced emits bubbles giving the positivity of the test (Hemraj et al. 2012). 

The importance of the oxidase test helps in the identification of the bacteria that has cytochrome C oxidase activity and finally the importance of the oxidation and fermentation reaction. The oxidative-fermentative (OF) test was first developed by the famous microbiologist Hugh and Leifson in the year of 1953. They developed OF media in order to differentiate between the oxidative bacteria (that is capable of producing acid from carbohydrates (glucose to pyruvic acid) under strict aerobic condition or supply of oxygen only) and fermentative bacteria (that is capable of producing acid both under aerobic and anaerobic conditions). The principle of PF test is the gram-negative rods metabolize glucose into pyruvate via fermentation or by aerobic respiration (oxidatively). During the anaerobic respiration, pyruvate is converted into weak acids (pyruvic acids) depending on the type of fermentation. This high concentration of acid produced as a result of the fermentation, turns the bromthymol blue indicator in the OF media from green to yellow.  The non-fermenting gram-negative bacteria metabolize glucose via aerobic respiration and thus produce weak acids during glycolysis and hence produce difference in colour. They also fail to survive in the absence of oxygen and fail to undergo fermentation (Bhuyar et al. 2012).

Results- General Observation

The process of fermentation is used in the food processing industry for converting carbohydrates into alcohol. Microorganisms such as yeast or bacteria that employ anaerobic respiration are used in this process. Widely consumed fermented foods that are processed by bacteria are vinegar, cheese, yoghurt. Escherichia coli are used for the production of ethanol, lactate, succinate and acetate (Förster, Andreas and Johannes et al. 2014).

Conclusion

Thus from the above study, it can be concluded that the unknown bacterial sample is Escherichia coli. The bacteria is gram negative and features rod shape. The bacterium is also show to give positive result in the oxidation/fermentation reaction.

There are several gaps in the identification study. In the future research procedure, there must be provision or steps to determine the external configuration of the bacteria with the help of the background staining or negative staining (Murray et al. 2015), determination of the flagella (live staining) and endospore (with the help of the endospore staining) (Mahon et al. 2014).

References              

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