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    Lab Report Food Microbiology

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    Joy Shaha
    The experiment involves preparing plates using nutrient agar medium to isolate and count viable microorganisms in a liquid sample using the plate method. A 24-hour E. coli culture is diluted in sterile water and spread on nutrient agar plates at different dilutions from 10-5 to 10-9. The plates are incubated at 30°C and colonies are later counted to determine the number of colony forming units per mL in the original sample. The standard plate count method allows quantification of actively growing bacterial cells and identifies the degree of microbial contamination in a food or liquid sample.

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    Lab Report Food Microbiology

    Uploaded by

    Joy Shaha
    30 views6 pages
    The experiment involves preparing plates using nutrient agar medium to isolate and count viable microorganisms in a liquid sample using the plate method. A 24-hour E. coli culture is diluted in sterile water and spread on nutrient agar plates at different dilutions from 10-5 to 10-9. The plates are incubated at 30°C and colonies are later counted to determine the number of colony forming units per mL in the original sample. The standard plate count method allows quantification of actively growing bacterial cells and identifies the degree of microbial contamination in a food or liquid sample.

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    lab report food microbiology

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    The experiment involves preparing plates using nutrient agar medium to isolate and count viable microorganisms in a liquid sample using the plate method. A 24-hour E. coli culture is diluted in sterile water and spread on nutrient agar plates at different dilutions from 10-5 to 10-9. The plates are incubated at 30°C and colonies are later counted to determine the number of colony forming units per mL in the original sample. The standard plate count method allows quantification of actively growing bacterial cells and identifies the degree of microbial contamination in a food or liquid sample.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    30 views6 pages

    Lab Report Food Microbiology

    Uploaded by

    Joy Shaha
    The experiment involves preparing plates using nutrient agar medium to isolate and count viable microorganisms in a liquid sample using the plate method. A 24-hour E. coli culture is diluted in sterile water and spread on nutrient agar plates at different dilutions from 10-5 to 10-9. The plates are incubated at 30°C and colonies are later counted to determine the number of colony forming units per mL in the original sample. The standard plate count method allows quantification of actively growing bacterial cells and identifies the degree of microbial contamination in a food or liquid sample.

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    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 6

    Experiment No:1

    Experiment Name:
    Plate preparation for total viable count with nutrient
    agar medium.
    Introduction:
    The plate method is a microbiological laboratory technique for isolating and
    counting the viable microorganisms present in a liquid sample, which is added
    along with or before molten agar medium prior to its solidification.This technique
    is generally used to count viable microorganisms in the given sample by
    enumerating the total number of colony-forming units (CFUs) within and/or on the
    surface of the solid medium. It is mostly used for enumerating bacteria; however,
    Actinobacteria, molds, and yeasts can also be isolated and enumerated.

    Objectives:
    The plate method is a microbiological laboratory technique for isolating and
    counting the viable microorganisms present in a liquid sample, which is added
    along with or before molten agar medium prior to its solidification.This technique
    is generally used to count viable microorganisms in the given sample by
    enumerating the total number of colony-forming units (CFUs) within and/or on the
    surface of the solid medium. It is mostly used for enumerating bacteria; however,
    Actinobacteria, molds, and yeasts can also be isolated and enumerated.

    Materials:
    I. 24 hours E.coli culture
    II. Nutrient agar plates
    III. Mac conkey agar plates
    IV. Nutrients broth
    V. Inoculating loop
    VI. Autoclave
    VII. Dessicator
    Procedure:
    A. Sample Dilution and Spread Plating
    1. Label one plate on the bottom for each of the following dilutions: 10 -5,10-6 , 10-
    7
    , 10-8,and 10-9.

    2. Label the dilution tubes as follows: label the two tubes (Navy blue caps)
    containing 9.9 mL sterile water as 10 -2 and 10-4; label the four tubes (Green caps)
    containing 9.0 mL sterile water as 10-5, 10-6, 10-7, and 10-8.

    3. Carefully and aseptically remove 0.1 mL (100µL) of the Serratia


    marcescens culture and pipette it into the tube marked 10-2. Mix the tube
    completely, being careful not to spill any of the contents. The vortex mixer will
    help you mix the contents of the tube.

    4. Change the pipette tip, and take 100µL (0.1 mL) from the 10-2 dilution and
    pipette it into the next 9.9 mL dilution tube marked 10 -4. Mix well. You have now
    prepared a successive 1/100 dilution, resulting in 10 -4 dilution of
    the original culture (1/100 multiplied by 1/100 = 1/10,000 = 10-4,

    which is the same as 10-2 x 10-2=10-4).

    5. Change your pipette tip again.

    6. Using a new pipette tip, transfer 100µL (0.1 mL) of the 10 -8 dilution onto the
    center of the agar surface of the plate marked 10-9.

    7. Spread plates: Using good aseptic technique, take one sterile blue L-shaped
    cell spreader out of the bag and reseal the bag. Starting with the plate marked 10 -9 ,
    using the spreader, gently push the liquid inoculum applied to the center of the
    plate, two or three times clockwise around the dish, then several times
    counterclockwise, turning the plate on the turn table as needed to
    obtain complete coverage.Continue with the rest of the spread plates using the
    same cell spreader. Make sure you are continuing to work backwards (from 10- 9 ,
    then 10-8 , then 10-7, then 10-6 etc.) from the most dilute suspension to the most
    concentrated suspension to minimize the amount of carryover from plate to plate.
    Again, use good aseptic technique, work quickly, do not touch the cell spreader on
    surfaces other than the agar plate you are using. If you accidentally “contaminate”
    the spreader by touching a surface (table or other), then dispose of it and get a new
    spreader. We are trying to minimize the use of the spreaders to reduce waste, but
    if it gets compromised, then replace it with a new sterile spreader. Dispose of the
    cell spreader in the small orange biohazard bin on your bench when you are done
    with your spread plates.

    Remember that the plates should be labeled as a ten-fold higher dilution than the
    dilution tube of the 0.1 mL sample being plated. For example, 0.1 mL of the 10 -
    6
    dilution tube should be plated on the agar plate marked 10-7.

    8.After the spread plating, leave plates agar side down for at least 30mins. in order
    for the inoculum to absorb onto the agar, then invert the plates and incubate at
    30°C.

    B. Counting colonies on plates


    1. Looking at your dilution plates prepared last period, choose the plates that have
    from 30-300 colonies on them. As this might take some practice in plate counting,
    you might need to choose all plates with what looks like a reasonable number of
    colonies to count.

    a. Those plates that have no microbial growth can be recorded as 0 or NG, No


    Growth.

    b. Those plates on which colonies are not individually distinct (their edges run
    together) can be recorded as TNTC, Too Numerous To Count.

    c. Those plates on which you cannot distinguish any individual colonies, the entire
    surface is covered with microbial growth, can be recorded as confluent.

    2.Count each colony to give a total colony count for each plate chosen. You will
    avoid counting a colony twice by marking off the colonies on the bottom of the
    plate as you count them. This requires, of course, that the plate be upside down. Be
    sure to count any small colonies. Record your results on the report sheet.

    C. Calculation of number viable cells/mL in the original sample


    • Choose the plate containing between 30 and 300 colonies.
    • Multiply the number of colonies on the plate by the final dilution factor. This
    gives the total viable cells/mL in the original sample.

    • Calculate the colony-forming units, CFU, per mL for the Serratia


    marcescens culture.

    Conclusion:
    The Standard Plate Count (SPC) means the colony count of the
    mesophilic bacteria growing under aerobic condition on standard
    methods agar (Plate Count Agar), and SPC becomes the representative
    index indicating the degree of the microbial contamination of the food.A
    viable cell count allows one to identify the number of actively
    growing/dividing cells in a sample. The plate count method or spread
    plate relies on bacteria growing a colony on a nutrient medium. The
    colony becomes visible to the naked eye and the number of colonies on a
    plate can be counted.

    References:
     https://bio.libretexts.org/Courses/North_Carolina_State_University/
    MB352_General_Microbiology_Laboratory_2021_(Lee)/
    05%3A_Enumeration_of_Bacteria/5.03%3A_Lab_Procedures-
    _Viable_Plate_count

     Sanders E. R. (2012). Aseptic laboratory techniques: plating methods.


    Journal of visualized experiments : JoVE, (63), e3064.
    https://doi.org/10.3791/3064

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