Microbiological Analysis: Lab Report

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Table of Contents

Purpose
Columbia CNA Agar
Eosin Methylene Blue Agar
Reference

Purpose

The purpose of this academic report is to summarize the results of a study of the cultivation of two cell cultures, Gram-positive S. epidermidis, and Gram-negative E. coli, on three nutrient media obtained in a previous experiment. This involved the study of cell morphology and colonial growth efficiency. In addition, microbiological analysis of the cultivation of the Unknown bacterial specimen on two new media, namely Columbia CNA Agar and Eosin Methylene Blue Agar, was performed.

Columbia CNA Agar

Columbia CNA Agar (or C-CNA for short) is a classic example of a selective media enriched with two antibiotics, colistin and nalidixic acid, and a defibrinated sheep’s blood (5%) as a cell differentiation factor for hemolysis studies. This type of medium is standardly used to isolate Gram-negative bacteria, with a source of nutrients in the form of carbon and nitrogen from the casein hydrolysate that is part of the C-CNA. The enriched nature of the nutrient medium creates unique opportunities for the growth of fastidious cultures: due to inhibition of growth properties of Gram-negative bacteria, C-CNA is selective against Gram-positive bacteria in the presence of whole blood. The selectivity is due to two sources of antimicrobial effect at once, namely colistin and nalidixic acid. The principle of action of both is based on the destruction of the cell wall of Gram-negative organisms and blocking the replication processes of bacterial DNA (Zhang et al., 2019). As a result, bacteria with a thin cell wall cannot be grown on C-CNA-type medium. If, after incubation time (usually 18-24 hours), the plate shows apparent growth in the form of a stroke, this indicates the ability of the microorganism to survive in the selective medium. Consequently, the bacterium is gram-positive. On the contrary, if the streak does not appear after a period of time, this signals the sensitivity of the strain to the action of the antibiotic. Consequently, the bacterium is gram-negative.

Eosin Methylene Blue Agar

Eosin Methylene Blue agar is the second nutrient medium studied in this study: it is both a selective and a differential medium for the isolation of fecal intestinal bacterial forms. EBM agar is a complex medium because it does not contain growth or suppression factors as such. At the same time, the Peptic digest of animal tissue is a source of both carbon and nitrogen necessary for bacterial growth. The selective nature of EBM is justified by the possibility of separating bacteria according to their metabolic ability to ferment lactose. The separation is realized through the use of the pigments Eosin and Methylene Blue, which act as indicators. In other words, Eosin Methylene Blue agar is effective against Gram-negative bacteria because the toxic effect of Methylene Blue tends to inhibit the growth of Gram-positive prokaryotes: this is due to the porous structure of the cell wall of such bacteria without a protective membrane. As a result of the differential nature, some bacteria have a dark blue or black coloration when grown. Hence, these are the ones that can ferment lactose. On the other hand, if bacterial colonies are grown but are not colored, this indicates that they cannot ferment lactose.

Reference

Zhang, Y., Wang, X., Li, X., Dong, L., Hu, X., Nie, T.,… & You, X. (2019). Synergistic effect of colistin combined with PFK-158 against colistin-resistant Enterobacteriaceae. Antimicrobial agents and chemotherapy, 63(7), e00271-19.

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Microbiological Analysis: Lab Report

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