Enumeration of Bacterial Cell Cultures and the Identification of an Unknown Bacterial Culture.

By Stephanie Waugh

Student Number: C3206138

Laboratory: Group 12, Tuesday 2-5:00pm

Abstract

This experiment was performed to use techniques common in microbiological laboratories, enumeration and identification tests. Absorbance measurements, pour plate CFU’s and spread plate CFU’s were documented at varying concentrations from an initial culture of Bacillus subtilis. A variety of tests were undertaken to identify an unknown bacterial sample, called Unknown 4. It was observed that the spread plate data was most useful in establishing the cells/mL of the samples, as it was more accurate than the pour plate data collected. After extensive testing, Unknown 4 was identified as Micrococcus luteus due to the results obtained and the morphology observed.

Introduction

It was hypothesised that the enumeration data would indicate a positive correlation between the number of cells/mL and the absorbance recorded by a spectrophotometer. It was also hypothesised that the use of a variety of tests on an unknown bacterial sample (Unknown 4) would lead to the identification of the sample.

Method

The procedure for this experiment was performed over the course of two weeks, with all methods using the aseptic technique. The experiment was divided into two distinct objectives, enumeration of a bacterial culture and the identification of an unknown bacterial sample.

Bacterial Enumeration

Using a bacterial culture of Bacillus subtilis a serial dilution was performed to obtain dilutions of 10-4, 10-6 and 10-8. By varying the aliquot volume, pour plates were created using 20mL of nutrient agar to achieve dilutions of 10-4 to 10-9. Dilutions of 10-5 and 10-7 were performed for the creation of spread plates with concentrations of 10-4-10-7. All plates were incubated at 37°C overnight before counting CFU’s present.

Identification of Unknown Sample

The sample used throughout this experiment was Unknown 4. Initial observations of colonies were recorded and a gram stain and hanging slide was prepared of Unknown 4 before performing metabolic profiling tests. A Eugon deep agar tube using the sample was prepared and was observed with the provided Eosin methylene blue and mannitol agar plates. The sample was then exposed to H2O2 and an oxidase strip to observe effects. The ability to ferment glucose was observed using the oxidation-fermentation (OF) test in conjunction with a sample in glucose fermentation broth with bromocresol purple.

Results

Enumeration of Bacillus subtilis cultures

A serial dilution of the stock culture of B. subtilis was performed to record the absorbance of the samples using a spectrophotometer.

Table 1. Average absorbance of serial dilution of B. subtilis at 600nm from class data, including standard deviation.

Concentration Average Absorbance Standard Deviation100% 0.8261 ±0.2341

50% 0.5107 ±0.1518

25% 0.2771 ±0.0889

12.5% 0.1383 ±0.0481

6.25% 0.0674 ±0.0345

Pour plates for B. subtilis at concentrations of 10-4 to 10-9 were generated and CFU’s in each plate were counted for analysis.

Table 2. Average CFU’s observed in B. subtilis cultures including standard deviation and indicating where CFU’s were too numerous to count (TNTC) or too few to count (TFTC).

Concentration CFU’s Standard Deviation10-4 TNTC N/A

10-5 TNTC N/A

10-6 112* N/A

10-7 75.1176 ±44.2788

10-8 TFTC N/A

10-9 TFTC N/A

* 112 CFU’s was the minimum number counted and was only obtained by one group. All other results were observed as TNTC

Spread plates were also created using the same initial B. subtilis culture in concentrations from 10-4 to 10-7.

Table 3. CFU’s observed on spread plates of B. subtilis including standard deviation, indicating where CFU’s were too numerous to count (TNTC) or too few to count (TFTC).

Concentration CFU’s Standard Deviation10-4 TNTC N/A

10-5 174.5 ±47.37615

10-6 59.3529 ±17.42463

10-7 TFTC N/A

From Table 2 and Table 3, it was observed that though the plates were at the same concentration theoretically, the number of CFU’s present was vastly different, in particular the plate 10-6 in both was found to have 112 and 59.3529 ± 17.42463 respectively. Due to the lower standard deviation indicated in the pour plates, the CFU value in plate 10-6 was used to determine the cells/mL, in relation to the absorbance of each sample.

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Figure 1. Graph generated in Excel 2013 demonstrating the relationship between the average cells/mL and the average absorbance, using error bars to depict the standard deviation of the results obtained.

From figure 1, it was observed that the number of cells/mL have a strong positive correlation, as indicated by the trendline.

Unknown 4 Observations

Initially, morphological observations were recorded. The culture of Unknown 4 provided was observed to be a dark mustard yellow colour, with small, pulminate colonies of a punctiform nature. A gram stain was performed on a sample of the culture, which when observed using a microscope, was observed to have round cells and to be gram positive, due to the dark purple

colour of the cells observed. From further observation on a hanging slide, it was found that the cells were non-motile, with only Brownian movement being observed. No spores were found.

Metabolic profiling of Unknown 4 was then performed to further narrow-down the possible bacteria. It was observed that Unknown 4 colonies did not form on the provided eosin methylene blue plate, whereas there was limited growth, but no colour change, on the provided mannitol agar plate. When a sample of Unknown 4 was cultured in a Eugon deep agar tube, it was observed that colonies only formed near the surface of the agar, indicating that the bacteria was aa obligate aerobe.

Unknown 4 was observed to give a positive result for oxidase activity, as the oxidase strip turned blue on contact with a colony. It was also found that when in contact with H2O2 colonies of the unknown produced bubbles, indicating a positive result for catalase activity.

Figure 2. Summary of results obtained from metabolic testing of Unknown 4 samples. Highlighted results indicate those observed.

Discussion

Enumeration of Bacillus subtilis cultures

From the results obtained, it was observed that the hypothesis was supported, as the number of cells/mL had a strong positive correlation with increasing absorbance when viewed in a spectrophotometer. It was also observed that pour plates and spread plates had a very different number of cells observed, with there being a significantly higher number of CFU’s in the pour plates, than there were in the spread plates. This is most notably indicated in the difference between the 10-6 plates from Tables 2 and 3, where the pour plate in table 2 was found to contain a minimum of 112, whereas the average number of CFU’s in the spread plate was 59.353 ± 17.42463.

Identification of Unknown 4

From the observations obtained in Figure 2, it was established from the identification chart that Unknown 4 was Micrococcus (Figure 3).

Figure 3. Cumulative results of the tests performed on Unknown 4 indicate that it is most likely Micrococcus.

Further analysis of the morphology of the Unknown 4 colonies concluded that it was highly likely to be Micrococcus luteus, due to the mustard yellow colour of the colonies (Perromat et al., 2003). The primary distinguishing factor between Micrococcus and Staphylococcus is the ability of Staphylococcus to grow in anaerobic conditions (Figure 3).

Potential Errors

The individual group results obtained varied somewhat from those obtained overall. Firstly, the Eugon deep agar tube for Unknown 4 was observed to have growth on the surface of the agar, but with the addition of pockets of air formed in the agar. This may have been caused by potential contamination by Escherichia coli from a previously used inoculating loop. Furthermore, the oxidase reaction strip was observed to only slightly have reacted with the colony it was exposed to, and was initially recorded as a negative result. This may have been due to insufficient exposure time to the colony, or a faulty oxidase strip.

References

Perromat, A., Melin, A. M., Lorin, C. and Deleris, G. (2003). Fourier transform IR spectroscopic appraisal of radiation damage in Micrococcus luteus. Biopolymers 72, 207-16.