Selective and Differential Medium Are Used to Isolate and Distinguish Bacteria from a Mixed Culture - Macconkey Agar Plate

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Introduction

Although bacteria might not be visible to the naked eye, they are in abundance in almost every environment on Earth. As development of detection techniques continue to improve over the last years, more studies are being conducted on bacterial species, structures and functions [1]. As bacteria form diverse communities, a singular bacterial species can prove difficult to be identified, cultured and isolated. As certain bacteria require specific nutrient and environmental conditions to grow, nutrient limitation or incubation environment can assist in obtaining a pure culture [2]. Supplemented growth media such as nutrient agar (NA) are usually also used in conjunction with aforementioned techniques, such as usage of differential mediums, enriched media and selective media.

Differential medium such as pH indicators and metabolic substrates are used to distinguish certain bacteria amongst all the growth present within the culture. Selective media are based on prevention of certain bacterial growth via inhibitors of metabolic pathways or implementation of antibiotics [3]. Enriched media contains additional nutrients to improve growth of organisms required for study.

An example of a selective and differential medium is MacConkey agar (MAC) which will be used in this experiment. Lactose fermenting bacteria can be distinguished from non-lactose fermenting bacteria on MacConkey agar [4] as lactose fermenting bacteria are easily distinguishable with their red, purple or pink appearance whereas non-lactose fermenting bacteria are colourless, translucent, or beige coloured. Addition of bile salts increases selectivity of this media as it inhibits certain growths. MacConkey agar is composed of 2% peptone, bile salts, lactose and a neutral red pH indicator.

Aim

In this experiment, selective and differential medium are used to isolate and distinguish bacteria from a mixed culture. A mixed broth culture is inoculated onto a nutrient agar plate followed by a MacConkey agar plate and growth is observed following inoculation.

Results

Table 1. Presence and appearance of bacterial growth on both NA and MAC plates

Table 2. Gram staining results from colonies obtained from Na and MAC plates

A sterile swab is used in aseptic conditions to obtain a sample of mixed broth culture of Bacillus megaterium (B. megaterium), Escherichia coli (E. coli) and Providencia rettgeri (P. rettgeri) by dipping the swab into the broth and then quickly inoculated onto the NA plate followed by the MAC plate. Both plates are then incubated at 37°C and observed after 24 hours for varying growths from different bacteria. Gram stains of colonies were performed at the end of the experiment to aid with identification of different bacterial species.

The results obtained from this experiment (Table 1) indicate that all bacteria from the mixed culture have shown some growth on the nutrient agar plate. B. megaterium showed spreading colonies whereas both E. coli and P. rettgeri may have outgrown. There is no growth of B. megaterium on the MAC plate, but both E. coli and P. rettgeri have shown positive growth. E. coli colonies appear pink in colour whereas P. rettgeri colonies appear beige in colour.

Gram stain of original mixture contained all three bacterial species, gram positive and negative rods. The final gram stain of separate colonies allow for more accurate detail to be determined, where B. megaterium are gram positive rods in chain arrangements, E. coli are gram negative rods in random arrangement and P. rettgeri are gram negative rods in random clusters (Table 2).

Discussion

The aim of this experiment is to isolate and distinguish certain bacteria from a given mixed broth culture of B. megaterium, E. coli and P. rettgeri. Two media types, nutrient agar (NA) and MacConkey agar (MAC) plates is used to aid in distinguishing different bacterial growth as well as help isolate these colonies for identification via gram stains.

A mixed culture broth of bacteria is inoculated first onto the NA plate then the MAC plate to prevent selective ingredients from the MAC plate transferring over to the NA plate, which will prevent growth of certain bacteria on the NA plate. The MAC plate is not incubated for longer than 24 hours to prevent overgrowth of possible contaminants before inoculation.