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Paul Pasco (1-22)Zuelay Rosario CruzGeneral Microbiology Laboratory (11:680:390)14 October 2014

Laboratory Report #1: Evaluating the Selectivity of GrowthMedia as a Function of pH — A Comparison of Mycophil and TSA

I. ABSTRACT:The purpose of this laboratory experiment was to demonstrate the phenomenon of

selection. This goal was accomplished by quantitative evaluation of the hypothesis that fungal and bacterial CFUs have a different growth pattern based upon the pH of the respective growth media used. In this experiment, the two growth media used were Mycophil and TSA. Data obtained revealed that fungal CFUs exhibit a distinct preference for media with a more acidic pH. In all of 8 data sets of 20-30 samples each, there was ≤45% more fungal growth on the more acidic medium. As a result, the conclusion that fungi grow optimally in a more acidic environment was made.II. INTRODUCTION:

In this laboratory experiment, the pervasiveness of microorganisms was utilized to demonstrate the selectivity, or lack thereof, of culture media. Microorganisms are present in the air, water, and soil. Not all types of microorganisms grow equally well under the same environmental conditions, however. For example, fungi prefer a more acidic pH, whereas bacteria prefer a more basic one (1). By exploiting this relatively simple preference, it is possible to choose, or select for, a predominance of either fungal or bacterial colony forming units (CFUs) by using an appropriately titrated culture medium.

To accomplish this goal, two types of culture media were used in this experiment. The first was tryptic soy agar (TSA), a general purpose culture medium with a pH of 7 that does not select for any particular type of microorganism. The second culture medium was Mycophil, a more selective type of culture medium with a pH of 5 that tends to select for fungi.

In this experiment, the hypothesis tested was that if the plate with the Mycophil medium contained a significantly larger proportion of fungi versus bacteria, the Mycophil medium had demonstrated the phenomenon of selection. The applications of supporting or rejecting such a hypothesis are significant. For example, determining the optimal pH range for fungal and bacterial growth, respectively, has innumerable applications in fields such as medicine, biotechnology, and plant science. Experimentally evaluating the current assumptions on fungal and bacterial growth as a function of pH with different types of growth media, and observing or failing to observe selection in the experiment, will add to a pragmatic body of knowledge usable by a broad spectrum of healthcare professionals, biotechnologists, and plant scientists.III. PROCEDURE:

Trypticase soy agar and Mycophil agar growth media were melted and stored in a water bath prior to the beginning of laboratory for use. The growth media were poured aseptically into sterile petri dishes, where the growth media were allowed to cool and harden as described in (2).

The prepared plates were inoculated with microorganisms outside the Department of Food Sciences Building, located at 65 Dudley Road on the Rutgers George H. Cook Campus in New Brunswick, New Jersey 08901. To accomplish the inoculation, the plates were placed

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directly underneath a bush located very close to the front door. This location was adjacent to the trash and recycling receptacles. The coordinates for the location where the two plates were inoculated are 40º28’45.0”N, 74º26’08.9”W. Photographs were also taken to include in the formal laboratory report for the experiment. The plates were opened at 13:25 on Tuesday, September 09, 2014. The plates were left open for a period of five minutes. Weather conditions at this time were cloudy, a temperature of 75ºF, 68% humidity, 61ºF dew point, and light and variable winds. There were no other weather conditions of note. After the five minute inoculation period was over, plates were immediately covered, taken back into the laboratory, and placed in plastic bins at room temperature. The plates were placed with agar side up to prevent any potential condensation from dispersing the CFUs. The plates were placed in an incubator for a period of one week by laboratory personnel.

During the next laboratory session, the plates were photographed by laboratory personnel. The macroscopic and microscopic morphological characteristics were examined by students with the unaided eye and a stereomicroscope at a magnification of 5X. Utilizing the assumption discussed in laboratory and the manual, round, shiny, and smooth colonies were considered bacterial, whereas filamentous colonies were considered fungal (2). This assumption was utilized uniformly to tabulate data for individual students and to pool data for all laboratory sections. These data may be viewed in Tables 1.1 and 1.2, respectively.

Figure 1.1: Location of TSA and Mycophil plates when inoculated outside Food Science Building.

This photograph shows the general location of where the plates were inoculated.

Figure 1.2: Location of TSA and Mycophil plates when inoculated outside Food Science Building.

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As compared to Figure 1.1, this photograph shows a closer view of where the plates were inoculated.

Figure 1.3: Location of TSA (left) and Mycophil (right) plates when inoculated outside Food Science Building.

As compared to Figure 1.2, this photograph shows a closer perspective of where the plates were inoculated. TSA plate is on the left; Mycophil plate is on the right.

Figure 1.4: Location of TSA (left) and Mycophil (right) plates when inoculated outside Food Science Building.

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As compared to the prior figures, this photograph shows an even closer view of where the plates were inoculated. TSA plate is on the left; Mycophil plate is on the right.

Figure 1.5: Location of TSA (left) and Mycophil (right) plates when inoculated outside Food Science Building.

As compared to the prior figures, this photograph shows a still closer view of where the plates were inoculated. TSA plate is on the left; Mycophil plate is on the right.

Figure 1.6: Location of TSA (left) and Mycophil (right) plates when inoculated outside Food Science Building.

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As compared to all the previous figures, this photograph shows the closest view of where the plates were inoculated. TSA plate is on the left; Mycophil plate is on the right.

IV. RESULTS:Table 1.1: Counts of fungal and bacterial CFUs on TSA and Mycophil growth media — individual data.

--- FUNGAL BACTERIAL% FUNGAL

(FUNGAL/FUNGAL + BACTERIAL)

MYCOPHIL (pH 5) 5 0 100TSA (pH 7) 10 9 52.6

The plates with Mycophil showed a uniform incidence of fungal CFUs only. The plates with TSA showed a more diversified distribution of both fungal and bacterial CFUs.

Table 1.2: Percentages of fungal and bacterial CFUs on TSA and Mycophil growth media — section data.

SECTION MYCOPHIL: % FUNGAL TSA: % FUNGAL WEATHER1 87 36 72ºF, 68% humidity2 87 19 69ºF, cloudy, not windy3 87 29 72ºF, sunny4 90 16 72ºF, partly cloudy5 90 21 80ºF, sunny6 90 32 84ºF, partly cloudy7 79 17 75ºF, sunny8 81 36 74ºF, partly cloudy

CLASS AVERAGE 86 26 ---These data, normalized for outliers by laboratory personnel, indicate that the plates with Mycophil for each section consistently contain a significantly higher percentage of fungal CFUs than the plates with TSA. In each section, with section 8 as the sole exception at 45%, each Mycophil plate contained at least 50% more fungal CFUs than the TSA plates in the same section.

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Figure 1.7: Photograph of TSA (left), Mycophil (right) plates exposed to nonsterile, outside air after 1 week of incubation.

The gross morphology of the microorganisms on each plate differed significantly. The plate with TSA medium is pictured on the left; the plate with Mycophil medium is pictured on the right.

The CFUs on the TSA plate were predominantly fungal (52.6%). The bacterial CFUs exhibited a circular form; were smaller than a dime in diameter; possessed smooth, glistening surface topography; a butyrous texture; cloudy coloration; a raised elevation; and entire margins. The fungal CFUs on the TSA plate displayed a circular form; were approximately the size of a dime in diameter; possessed irregular surface topography; a dry texture; cloudy coloration with occasional green pigmentation; flat elevation; and filiform margins.

The CFUs on the Mycophil plate were exclusively fungal. These CFUs exhibited a circular form; the diameter of a quarter or greater; irregular surface topography; dry texture; cloudy or iridescent coloration; flat or umbonate elevation; and filiform margins.

The fungal colonies on the Mycophil plate were more elevated than the fungal colonies and bacterial colonies on the TSA plate. The fungal colonies on the Mycophil plate also exhibited a broader range of coloration than the fungal and bacterial colonies on the TSA plate. The fungal colonies on the TSA plate were predominantly white, with a small percentage of green pigmentation. In contrast, the fungal colonies on the Mycophil plate were predominantly green, with a less extensive percentage of green pigmentation. There was also a notable cluster of what resembled iridescent “crystals” of pigment. These crystals appeared mainly pale yellow or orange.

V. DISCUSSION:As indicated in Tables 1.1 and 1.2, as well as Figure 1.7, there is an exclusivity of

fungal CFUs on the plate with Mycophil culture. In comparison, there is a more diversified growth of CFUs on the plate with TSA culture. These data support the tested hypothesis. That is, these data support the conclusion that Mycophil is a selective medium, which strongly encourages the growth of fungi, whereas TSA is a general purpose medium, which means it is unselective and does not encourage the growth of any particular type of CFU. Therefore, the phenomenon of selection was demonstrated by this experiment. These

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conclusions are in alignment with the predictions that were formulated prior to conducting the experiment.

In addition, this hypothesis is consistent with the established patterns in the literature. Bacteria are presumed to possess a narrower optimal pH range than fungi (3). This finding may explain why there were fungal CFUs on both the TSA and Mycophil plates, but bacterial CFUs only on the plate with TSA. The potential ability of fungi to encompass a broader pH range would be consistent with the experimental outcome observed. More research to evaluate this phenomenon is recommended.

VI. REFERENCES:Waksman, S.A. 1921. A Method for Counting the Number of Fungi in the Soil. Journal of Bacteriology, Volume VII, No.3; pp. 339-41.

Davis, D.F. 2014. Exercise 2: Ubiquity — Selection of Fungi or Bacteria from Air., p. 13-14. In D.F. Davis and I. Rauschenbach (ed.), General Microbiology: 680:390. Laboratory Exercises Fall 14.

Rousk, et al. 2010. Soil bacterial and fungal communities across a pH gradient in an arable soil. Multidisciplinary Journal of Microbial Ecology, Volume 4; pp. 1340-51.

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