lab #9 introduction materials & methods · kimberly tierney microbiology lab lab 9 performed...

Kimberly Tierney Microbiology Lab Lab 9 Performed 4/11/2013 Instructor: Travis David

Lab #9

Introduction Food-borne illness is largely caused by the presence of bacteria in red meat. However, much of these

harmful bacteria can be destroyed and prevented by sanitation and safe cooking practices. This lab will

establish the presence of microbes in variations of cooking practices and evaluate the effective

sanitation of the cutting board utilized.

Materials & Methods Experiment: Microbial Analysis of Meat

Class samples:

1. Fresh – medium rare

2. 2 day old – medium rare

3. Expired - well

4. Expired – medium rare

Weigh 1 gram of beef meat from samples provided, place raw meat on specified and labeled side of

cutting board, cook 2 day old meat to medium rare, place cooked meat on opposing labeled side of

cutting board.

Part 1: Cooked Meat Analysis

Place 1 gram cooked meat, broken into pieces, into test tube of the original dilution (10⁰) with 10 mL of

MRD solution and shake to mix. Place 9.9 mL MRD in following 5 test tubes to produce serial dilutions of

10⁻², 10⁻⁴, 10⁻⁶, 10⁻⁸, and 10⁻¹⁰ by transferring 0.1 mL from original (or previous dilution tube to

inoculated) via a 20 – 200 µL pipette. Using the spread plate method, 1 mL is transferred from

correlating dilution to labeled plates as follows:

Spread Plates of 2 Day Old Meat Cooked to Medium-Rare

Medium Dilutions

NA 10⁻² 10⁻⁴ 10⁻⁶ 10⁻⁸ 10⁻¹⁰

EMB 10⁻² 10⁻⁴ 10⁻⁶ 10⁻⁸

BP 10⁻² 10⁻⁴ 10⁻⁶

MYP 10⁻² 10⁻⁴ 10⁻⁶

MRS 10⁻² 10⁻⁴ 10⁻⁶ 10⁻⁸

Part 2: Cutting Board Analysis

Wash cutting board, take a sterile swab moistened with MRD of the cutting board from labeled raw to

cooked side. Place swab of potential cutting board microbes into a test tube containing 1 mL of MRD as

Kimberly Tierney Microbiology Lab Lab 9 Performed 4/11/2013 Instructor: Travis David the original (10⁰) test tube in the series of serial dilutions 10⁻¹, 10⁻², and 10⁻³. The following 3 test tubes

of the serial dilution contain 9 mL MRD, and 1 mL transferred from previous dilution test tube. Using the

spread plate inoculation method, the following dilution plates were prepared with 1 mL from

corresponding dilution test tubes:

Spread Plates of Cutting Board Swab from Raw to Cooked Meat

Medium Dilutions

NA 10⁻¹ 10⁻² 10⁻³

VRBG 10⁻¹ 10⁻²

Part 3: API Test

An API test was performed on a light pink microbial colony retrieved from the VRBG spread plate cutting

board swab with a dilution of 10⁻¹. The procedure followed to perform an API test is described

previously in lab 7 under exercise 5-29.

API Reagent synonyms & description:

Ferric chloride Dropper bottles (unlabeled)

Sulfonic acids Nitrate A

N,N dimethyl - 1 - napthylamine Nitrate B

Results Part 1: Cooked Meat Analysis

Spread Plates of 2 Day Old Meat Cooked to Medium-Rare

EMB Spread Plates:

Dilution 10⁻² Dilution 10⁻⁴


BP Spread Plates:

MYP Spread Plates:

Dilution 10⁻⁶ Dilution 10⁻⁸

Dilution 10⁻² Dilution 10⁻⁴ Dilution 10⁻⁶


Kimberly Tierney Microbiology Lab Lab 9 Performed 4/11/2013 Instructor: Travis David MRS Spread Plates:

NA Spread Plates:

Dilution 10⁻² Dilution 10⁻⁴

Dilution 10⁻⁶ Dilution 10⁻⁸




Spread Plates of Cutting Board Swab from Raw to Cooked Meat

VRBG Spread Plates:

NA Spread Plates:

Dilution 10⁻⁸ Dilution 10⁻¹⁰

Dilution 10⁻¹ Dilution 10⁻²

Dilution 10⁻¹ Dilution 10⁻² Dilution 10⁻³


Class Plate Counts:

Group 1 Group 2 Group 3 Group 4

Meat/ Cooked

Fresh - Medium Rare

2 Day - Well Done

2 Day - Medium Rare

Expired- Medium Rare

Medium Dilution Plate

CFU Dilution Plate

CFU Dilution Plate

CFU Dilution Plate

CFU

MYP 10⁻² 194 NG NG 10⁻⁶ 71 10⁻² 2

BP NG NG NG NG NG NG NG NG

EMB 10⁻⁸ 134 NG NG 10⁻⁴ 133 10⁻² 38

MRS 10⁻⁶ 45 NG NG 10⁻⁴ 205 10⁻² 36

NA Not Countable

Not Countable

NG NG 10⁻⁶ 226 10⁻² 126

Swab Wood Wood Plastic Plastic

NA NG NG 10⁻¹ 123 Not Countable

Not Countable

10⁻¹ 24

VRBG NG NG NG NG Not Countable

Not Countable

10⁻¹ 11

NG = no growth

Part 3: API Test

Part 1: Initial Spontaneous Test Results

Part 2: Reagent Test Results


Discussions The following selective and differentiating mediums are utilized to try to identify which microbes are

present and at what quantity. The following table describes which observations may occur within

various types of medium and the common micro-organisms expected to grow. This table will be utilized

as a reference to attempt to identify growth on cultured spread plates. All information was retrieved

from http://www.neogen.com/Acumedia.

Medium Agar Name

Used For Possible Observations

Expected Micro-organisms

NA Nutrient Agar

cultivation of a wide variety of microorganisms

N/A Bacillus subtilis

Escherichia coli

Salmonella typhimurium

Staphylococcus aureus

Streptococcus pneumoniae

Streptococcus pyogenes

EMB Refer to previous lab (Lab # 8) BP

MYP

MRS deMan, Rogosa, Sharpe

the cultivation of lactobacilli

N/A Lactobacillus casei

Lactobacillus fermentum

Lactobacillus plantarum

VRBG Violet Red Bile Glucose

enumeration of Enterobacteriaceae in foods

Dextrose fermenters produce red

Enterobacter aerogenes

Pink colonies w/red precipitate

Escherichia coli Pink colonies w/red


colonies with red-purple halos in the presence of Neutral Red, the pH indicator

precipitate

Pseudomonas aeruginosa

Colorless to grey

Salmonella typhimurium

Pink colonies w/red Precipitate

Staphylococcus aureus

N/A

*Per the lab syllabus, MRS should grow lactobacilli, and VRBG should grow enterobacteria.

Part 1: Cooked Meat Analysis (2 Day Old – Cooked to Medium Rare)

EMB Spread Plates:

The EMB spread plates displayed dense growth, appearing colorless (possibly Pseudomonas aeruginosa

or Salmonella typhimurium) covering about 90% of the agar surface with little isolation achieved and

few colonies. Growth was uniform within the thick, blanketed areas. The micro-organism likely performs

aerobic respiration. The density of growth and amount of surface area displaying growth decreased as

the dilution factor increased. Due to the affluent growth, a result can be deduced that EMB supplies

nutrients necessary for the growth and isolation of a single species of microbe present in the sample of

meat cooked to medium rare.

BP Spread Plates:

The only BP spread plate to successfully produce growth was a dilution of 10⁻² in which the total number

of colonies was too few to count with very small diameter and large spacing between colonies. Growth

appears black, round and raised (possibly Enterococcus faecalis or Staphylococcus epidermidis). Due to

the lack of growth produced upon the BP spread plates, an interpretation can be deduced that BP plates

do not provide the necessary nutrients nor has an inhibitory effect upon the microbes found within the

cooked meat sample.

MYP Spread Plates:

The MYP spread plates displayed little colony isolation but rather densely swarming growth of a

colorless (possibly Bacillus subtilis) microbe upon the surface area of all dilutions prepared. On one

occasion (10⁻⁴), the color of ¼ of the agar is observed to have undergone a reaction changing from

yellow-orange to red-orange. The microbial growth pattern denotes aerobic respiration. Although

Bacillus cereus typically produces pink microbial growth, it may also create a white precipitate from the

reaction between lecithinase and the egg yolk within the medium; there exists a white precipitate across

the surfaces of dilution plates 10⁻² and 10⁻⁴.

MRS Spread Plates:

The MRS plates confirm the presence of lactobacilli within the sample of meat cooked to medium rare

by producing microbial growth upon all four dilution plates prepared. MRS plates are utilized for the

cultivation of lactobacilli specific micro-organisms; however it is possible to demonstrate the presence

of differing types of lactobacilli. The growth upon the MRS plates appeared as isolated opaque white

rounded and raised small colonies, as well as a more translucent white branching growth. Total cell

Kimberly Tierney Microbiology Lab Lab 9 Performed 4/11/2013 Instructor: Travis David growth decreased as the dilution factor was increased, but total growth upon the first dilution plate of

10⁻², was too great to be counted.

NA Spread Plates:

Nutrient agar is utilized to promote the growth of many various micro-organisms rather than inhibiting

or limiting specific organisms. The five NA plates produced displayed large variations of growth, possibly

indicating a mistake in the dilution process due to a lack of growth upon the first dilution plate which

should contain the largest quantity of bacteria; however growth of colonies too numerable to count

upon the second dilution spread plate. Colonies upon dilution plate 10⁻⁴ appeared with differing small

diameters of symmetrical off-white demonstrating a preference of spacing rather than dense clusters, as

well as a preference for aerobic metabolism demonstrated by the location at the surface of the medium.

The growth upon dilution 10⁻⁶ and 10⁻¹⁰ appear as thin, translucent film densely swarming rather than

preferring spaced colonies.


VRBG Spread Plates:

The microbial growth upon the VRBG spread plates demonstrate definitively that the sanitation

methods performed do not prevent or destroy micro-organisms upon a plastic cutting board. The

variation of observed physical traits in growth, including color, shape, both absence and presence of

halos and changes to color or reactions with the agar medium may indicate differing species. The VRBG

exhibits a large shift in color from a deep opaque maroon to a transparent peach following reactions and

nutrient depletion by the large white (surrounded in grey halo) and pink isolated colonies. VRBG agar is

utilized to present enterobacteria; however growth present may be Enterobacter aerogenes, Escherichia

coli, Pseudomonas aeruginosa, Salmonella typhimurium, or Staphylococcus aureus when compared to

traits presented in observational growth described in the above table.

NA Spread Plates:

The NA spread plates displayed little to no growth. The growth which exhibited upon the agar displayed

3 distinct variations in growth patterns. Upon the 10⁻¹ dilution plate, growth appears as extremely small

densely clustered off-white colonies or a thin film. Upon the 10⁻² dilution plate, 2 single moderately

sized tan colonies appear at one end of the agar, and at the other a growth appears with the similarity of

a dried, pressed off-white flower. The result of little to no growth is perplexing due to the objective of

nutrient agar to support a wide variety of growth and nutrient needs.

Class Plate Counts:

The results of the class plate counts performed upon the variations of fresh to expired meat cooked to

either well done or medium rare are difficult to analyze due to inconsistency of growth. It would have

been expected that medium-rare meat would display greater presence of microbe than well-done meat

because the extended duration and exposure to heat should destroy more bacteria. Also, it would be

expected that expired meat would demonstrate a larger presence of bacteria, unless all nutrients were

depleted resulting in cell death.

Kimberly Tierney Microbiology Lab Lab 9 Performed 4/11/2013 Instructor: Travis David The results depicted by the plate counts performed upon wooden and plastic cutting boards can be

interpreted as plastic cutting boards enabling greater growth of bacteria, possibly due to cuts made by

knives into the plastic which can harbor growth. The type of wood used may have been antimicrobial or

a type which will inhibit the growth of bacteria resulting in no growth upon dilution plates.

Part 3: API Test

The API test performed upon a colony selected from the VRBG swab dilution plate, demonstrated many

positive spontaneous reactions, as well as many reactions to reagents. However, identification could not

be made. Attempts were made to input variations of the seven digit code where possible false positive

results could have been interpreted, still identification was not possible.

API Results & Interpretations

Spontaneous Reagent

Test Observation Result Observation Result Interpretation Symbol

ONPG Off-white Positive Produces Beta-galactosidase

+

ADH Pink Positive Produces arginine dehydrolase

+

LDC Pink Positive Produces lysine decarboxylase

+

ODC Red Positive Produce ornithine decarboxylase

+

CIT Blue Positive Utilizes citrate as sole carbon source

+

H₂S Clear Negative Does not reduce sulfur -

URE Yellow Negative Does not produce urease -

TDA Clear Negative Light peach Negative Does not produce tryptophan deaminase

-

IND Clear Negative Yellow Negative Does not produce indole -

VP Clear Negative Pink Positive (possible contaminant)

Produces acetoin +/-

GEL Black dispersed

Positive Produces gelatinase +

GLU Blue Negative Yellow Positive Ferments glucose +

MAN Blue Negative Does not ferment mannitol

-

INO Yellow Positive Ferments inositol +

SOR Blue Negative Does not ferment sorbitol -

RHA Blue Negative Does not ferment rhamnose

-

SAC Blue Negative Does not ferment sucrose -

MEL Blue Negative Does not ferment melibiose

-

AMY Yellow Positive Ferments amygdalin +

ARA Blue Negative Does not ferment -


arabinose

Key

Spontaneous reactions occurred

Reagents Added

Variations of seven digit code: 7307201, 7306201

lab #9 introduction materials & methods · kimberly tierney microbiology lab lab 9 performed...

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