mjohnson mmbb417 report 9

8/2/2019 MJohnson MMBB417 Report 9

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Mark Johnson

9/20

Microbiology of Fresh Fruits and Vegetables

Overall Objective: The overall objective of this lab was to explore the common bacteriafound on whole fruits and vegetables, and microscopically observe fungi.

Objectives: One objective of this lab was to examine the microbial biota of the interior and exterior of fruits and vegetables both before and after sanitation. The bacteria will beobserved using several selective and non-selective medias. Another objective of this labwas learn to use a simple stain to observe fungi microscopically.

Materials and Methods: We were provided two melons to serve as examples. First, we

took a sample of the outer portion of one melon, stomached it, and diluted it to 10-6

. Weanalyzed each step-wise dilution using Standard Methods Agar (SMA), Potato DextroseAgar (PDA) Dichloran Rose Bengal Chloramphenicol Agar (DRBC), 3M Petrifilm Yeastand Mold Plates, and SimPlate Yeast and Mold CI. Next, we sanitized the outside of other melon and took a sample from the interior of the fruit. We analyzed this sample using thesame media used previously except for the 3M Petrifilm and Simplate. After incubation,we took isolates from the PDA and DRBC plates and streaked them on Mycophil Agar andMalt Extract Agar. After these plates were incubated, we observed the coloniesmicroscopically using a fungal stain.


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Results:

Table 1. Counts:10 -1 10 -2 10 -3 10 -4 10 -5 10 -6

Plate 1 / 2 Plate 1 / 2 Plate 1 / 2 Plate 1 / 2 Plate 1 / 2 Plate 1 / 2

SMA TNTC/TNTC 70/67 5/11 0/0 1/0 0/0

PDATNTC/TNTC

TNTC/TNTC

TNTC/TNTC

TNTC/0

TNTC/4

0/0

DRBC64/81

8/5

1/1

0/0

0/0

0/0

3M

PetrifilmYeast and

Mold

41/46

6/7

0/1

0/0

0/0

0/0

SimPlateYeast andMold CI

PositiveWells :All/All

Population :>738/>738

PositiveWells:All/14

Population :>738/

30

PositiveWells:

38/12

Population :100/26

PositiveWells :

14/18

Population :30/40

PositiveWells :

36/All

Population :94/

>738

NotPlated

TNTC stands for too numerous to count.


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Table 2. Colony Morphology Observations:Bacteria Yeast Molds

SMA-round, convex,smooth margin,~1mm, white,

translucent-rhizoid colony, palewhite, ~1-2mm-round, yellow,smooth, raisedcolonies

-None -None

PDA-yellow, convex,smooth, clustered

-white, fuzzy, roundw/ radiating margins

-green, fuzzy, droplike shape, small

DRBC-pink, convex,yellow center

-white, fuzzy, round,filamentous,

scalloped, large

-green, fuzzy, round

3M Petrifilm Yeastand Mold

-light blue/ black spots-blue-green dots-3 black splotches,webbed

-teal dots -1 black, webbedsplotch

SimPlate Yeast andMold CI

N/A N/A N/A

Table 3. Counts:10 -1 10 -2 10 -3 10 -4 10 -5 10 -6

Plate 1 / 2 Plate 1 / 2 Plate 1 / 2 Plate 1 / 2 Plate 1 / 2 Plate 1 / 2

SMA0/1

0/0

0/0

0/0

0/0

1/0

PDA0/0

0/0

0/0

0/0

0/0

0/0

DRBC0/0

0/0

0/0

0/0

0/0

0/0


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Table 4. Colony Morphology Observations:Bacteria Yeast Molds

SMA-few filamentous fungi-spreader colonies

-None -None

PDA -No growth -No growth -No growthDRBC -No growth -No growth -No growth

Table 5. Observations:ColonyMorphologyObservations

MicroscopicObservations(low power)

MicroscopicObservations(high power)

Mycophil AgarIsolate 1

-white, filamentous, branchine, convex,white w/ slight greencenter

-blue, filamentous,~5m, 40X, similar to aspergillus

-Yeast, looked likefilaments, stringy


-white, filamentous,convex, branchine

-blue, rods with bulbous heads,~3m

-long rod, bulboushead, blue, >50m inlength, resembledsyncephalastrum


-white, filamentous,Branchine, convex

-white branches,slightly blue, ~3mstem, ~1m hyphae

-long stem w/ branching apex-segmentation of hyphae

Malt Extract AgarIsolate 1

-white, filamentous,raised, hair-like

border

-thick stem leadingto thin branchinghyphae, free spores

-very long stem, branching hyphae,spores, resembled

paecilomyces


-white, filamentous,hair-like, hilly

-thin, curving,spreading filaments,free spores

-many overlapping branching hyphae,filamentous,numerous spores,resembledgeotrichum


-white, filamentous, branchine, opaque

-filamentous,spreading hyphae,few spores

-on ends of stems-conidia- rush-like,some spores,resembled

penicillium


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Discussion: This lab was significant in that it introduced us to the micro biota that could be present on the interior and exterior of whole fruits and vegetables. We used severaldifferent enumeration methods to observe bacteria, molds, and yeast that can be found in or

on these food products. We explored to how select for the different kinds of microbes(bacteria, yeast, or mold) that can be found on these products. This lab also introduced usto the fungal stain and observing fungi microscopically.

The several methods we used to observe microbes on the outer portion of the melon produced various results (Tables 1 & 2). When using SMA, we successfully obtainedgrowth. This media is non-selective, and we found bacteria, yeast, and mold growing on itafter incubation. The 10 -2 and 10 -3 dilutions produced the best results in terms of acceptableamounts of colonies.

The PDA showed growth as well, but in most cases, there were too numerous of colonies to count successfully. This media is selective for yeasts and molds, but we

observed bacterial growth on them as well as yeast and molds.The DRBC is selective for yeast and mold, but also grew bacteria in our experiment. The dilutions of 10 -1-10 -3 gave acceptable results, whereas no growth appearedwith the other dilutions.

The 3M Petrifilm Yeast and Mold media showed growth in the same dilutions asDRBC. It gave us acceptable, countable numbers of colonies, but one would be unable todistinguish between yeast and molds.

The SimPlate Yeast and Mold media successfully showed growth, but the resultswere sporadic. The 10 -3 and 10 -4 dilutions gave the most clear and concise results, whereasthe others sometimes showed completely positive results in one tray and only a few

positives in the duplicate.Using these methods, one could further isolate and identify the microbes in order to

fully examine the safety of the food.

The enumeration methods we used to examine the inner sample of the melon gavemuch less growth (Tables 3 & 4). This may have been related to the sanitization of themelon before acquiring the sample. Only a few microbes successfully grew on the non-selective SMA. There was no growth at all on the PDA and DRBC plates.

We had to use isolates from the outer melon sample as supposed yeast or moldcolonies to further examine on the mycophil and malt extract medias. We successfullyobtained three separate isolates from each media to examine using a fungal stain (Table 5).

Conclusion: This lab was very successful in introducing us to the microbes possibly present in/on fruits and vegetables. I had previously not considered the fact that they could be present on these food products. I learned that these food products could very possiblycontain microbes and the importance of washing them before consumption. I learned of the different techniques to select for various microbes that could be present. This includesseveral medias which select for yeasts and molds.


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When plating the inner portion of the melon on SMA, we received unclear results.We were enumerating for bacteria, but it seemed only fungi/ spreader colonies grew. Thismay have been due to improper spreading. Next time I would be more careful in themixing of the agar and sample.

When using the SimPlate, the results were rather erratic, which maybe have beendue to a failure to drain the excess liquid after inoculation. This extra liquid producedunusual results sometimes. In the future, care will be taken to remove this extra liquid.

References:

Unlu, Gulhan. Fall 2011. Food Microbiology: Laboratory Manual for FS 417 & MMBB417, p. 4-31. University of Idaho, I.D.

mjohnson mmbb417 report 9

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