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FOOD MICROBIOLOGY WORKSHOP An Introduction to Microorganisms in Foods

June 6 June 9, 2011

Food Science and Technology Program University of Puerto Rico-Mayaguez Campus Sponsored by USDA-NIFA-HSI Grant PRE 2010-02088

Exercise 1. Introduction to the Food Microbiology Laboratory


USDA-NIFA-HSI Grant PRE 2010-02088

Laboratory Safety Rules: o These are rules for your own safety: Laboratory coat and closed shoes (no sandals) are required in the laboratory. No eating or drinking is allowed in the laboratory. No mouth pipetting is allowed. Wastes are to be deposited in identified containers.

Beneficial and detrimental aspects of microorganisms in foods: students will learn the importance of beneficial microorganisms in fermented food products such as: Yogurt Sauerkraut Mab

o Detrimental aspects of microorganisms responsible for the shelf-life of products such as: Fresh milk Vegetables Meats cross contamination and

Introduction to two basic concepts in food microbiology: temperature abuse.

o Cross contamination: Students will use Petri plates with Tryptic Soy Agar (TSA) as to demonstrate the presence of microorganisms in the environment. Each student will be provided with a Petri plate containing TSA. Students will expose their plate in a selected area within the building (laboratory table, elevator, restroom, and classroom). Plates will be incubated for 24-48 h at 35C and plates will be observed for colony diversity.

USDA-NIFA-HSI Grant PRE 2010-02088

o Temperature abuse: Students will observe the consequences of exposing perishable foods (milk, meat) to temperature above that recommended for their storage. Spoiled milk and spoiled meat will be used as examples of the effects of temperature abuse on the organoleptic characteristics of perishable products.

Intrinsic and extrinsic factors that affect the life and death of microorganisms in foods. Students will learn about : o intrinsic factors: aw, pH, nutrient content, and antimicrobial constituents o extrinsic factors: gaseous environment and temperature

Sampling foods for microbial analysis. o Students will learn how to homogenize a solid food sample using a stomacher while at the same time they will be introduced to the concept of serial dilutions (Figures 1 and 2). Weigh 25 g of clams in a stomacher bag containing 225 ml of Alkaline Peptone Water (APW). Place the bag in the stomacher for one minute. Serial dilutions of the sample will be done for the isolation of vibrios, following the Bacteriological Analytical Manual (BAM). Transfer 10 ml to 90 ml APW dilution bottles to reach up to a 10-4 dilution. Incubate the bag and the dilution bottles overnight at 35C.

USDA-NIFA-HSI Grant PRE 2010-02088

Figure 1: Homogenization of solid food samples.

USDA-NIFA-HSI Grant PRE 2010-02088

25 g

225 ml buffer

10 m l from hom og enized sam ple

10 ml

10 ml

10 ml

1m l from hom og enized sam ple

90 m l

90 m l

90m l 90 m l

90m l 90 m l

S tom acher

1 ml

1 ml

1 ml

1 ml

Melted ag a

10 -1

10 -2

10 -3

10 -4

10 -5

Figure 2: Principle of Serial Dilutions.

USDA-NIFA-HSI Grant PRE 2010-02088

1m l

1m l

1m l

9m l

9m l

9m l

S am ple

10 -1

10 -2

10-3

1m l

1m l

1m l

Melted a g ar

S wirl plates g ently while ag ar isstill in liquid state. Incubate at proper tem perature.

Use platesthat have between 25-250colonies .

USDA-NIFA-HSI Grant PRE 2010-02088

Exercise 2. Isolation of microorganisms from food samples Students will isolate three microorganisms of importance in food microbiology, using selective media. o Students will learn how to select microbiological media in order to isolate different microorganisms of interest in foods. o The difference between selective and differential media will be discussed.

Working in groups, students will isolate the following microorganisms using media specified in the BAM: o Vibrios. The homogenized incubated clam sample from Exercise 1, will be used to isolate vibrios using Thiosulfate Citrate Bile Sucrose Agar (TCBS), a selective medium for the isolation of vibrios. Students will prepare serial dilutions of the sample following the diagram in Figure 2. APW will be used as the dilution media and bottles will be incubated overnight at 35C. The following day students will streak TCBS plates using growth from the incubated bottles. Plates will be incubated overnight at 35C. Colonies on the incubated plates will be observed (non-sucrose fermenters such as Vibrio parahaemolyticus are green).

o Coliform bacteria: Violet Red Bile Agar will be used for the isolation of coliform bacteria from two varieties of local fresh cheese (queso del pas and queso de hoja). Students will homogenize cheese samples and carry serial dilutions. Samples of 1ml and 0.1 ml will be plated following the pour plate technique.

USDA-NIFA-HSI Grant PRE 2010-02088

Plates will be incubated overnight and those with 25-250 colonies will be selected for determining CFU/ml. A discussion on the significance of coliform bacteria in processed products will follow.

o Staphylococcus aureus: Baird Parker (BP) Agar will be used for the isolation of S. aureus from two varieties of local fresh cheese (queso del pas and queso de hoja). Homogenized samples used for the isolation of coliforms will also be used to streak plates of BP Agar. One ml of homogenate will be distributed in three plates of BP Agar (0.3, 0.3, and 0.4 ml) using a streaking rod. After drying, plates will be incubated for 24 h at 35C. Colonies representative of Staphylococcus aureus (black with an outer clear zone) will be counted. The significance of S. aureus in processed foods will be discussed.

Exercise 3.

A. Survival and/or growth of microorganisms considering the following parameters: pH, temperature, and salt. Students will determine the pH of: milk, carbonated water, Coca-Cola, orange juice, pickles, and olives. Microbial stability of these products will be discussed with emphasis on the effect of pH on microorganisms. o In order to illustrate the effect of pH on the growth of microorganisms, two microorganisms will be used: Escherichia coli and Saccharomyces cerevisiae. Two sets of three tubes of Tryptic Soy Broth (TSB) with pH of 3, 7, and 10, respectively, will be inoculated with each microorganism. Tubes will be incubated at 37C for 24 hours. An extra set of TSB tubes will be used as a control.

USDA-NIFA-HSI Grant PRE 2010-02088

Turbidity will be visually determined in each tube after incubation. A discussion on the use of pH for the control of microorganisms in foods will follow.

In order to illustrate the effect of temperature on microbial growth, four TSA Petri plates will be divided in half and inoculated with Pseudomonas aeruginosa on one side and Escherichia coli on the other. o Plates will be incubated at 4C, 25C, 37C and 55C for 24 hours. Plates will be examined for growth. o A discussion on the use of temperature for the control of microorganisms in foods will follow.

In order to illustrate the effect of salt on microbial growth in foods, Buffered Peptone Water (BPW) with different concentrations of NaCl will be used. o Sets of BPW tubes containing 0%, 7%, and 10% NaCl will be inoculated with Escherichia coli, Staphylococcus aureus and a halophilic microorganism, respectively. o Tubes will be incubated at 37C for 24 hours. An extra set of tubes will be used as a control. o Turbidity will be visually determined in each tube after incubation. o A discussion on the use of salt for the control of microorganisms in foods will follow.

B. The effect of sanitizing agents on microorganisms Each group of students will be provided with plates containing TSA. Each plate will be divided in six (6) segments labelled as: Control, 0 min, 30 sec, 1 min, 3 min, and 5 min. Escherichia coli will be used for a demonstration on the effect of sanitizing agents using different contact times. Sodium hypochlorite (50 ppm), Lysol, and hand sanitizer will be tested as sanitizing agents. o Tubes of Tryptic Soy Broth (TSB) containing a fresh culture of E. coli will be provided to each group of students.

USDA-NIFA-HSI Grant PRE 2010-02088

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Students will inoculate the tubes containing the sanitizing agents with 0.5 ml of the bacterial suspension and immediately will inoculate the TSA plate using a loop (0 minutes).

o Contact time between the microorganism and each sanitizing agent will be allowed to take place (1 min, 3min, and 5 min), inoculating the TSA plates at the end of each period (Figure 3).

USDA-NIFA-HSI Grant PRE 2010-02088

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Figure 3: Exercise to determine the effect of sanitizing agents in microbial growth.

C. Differential staining technique the Gram Stain The Gram stain is a differential stain that allows for a rapid distinction between two major groups of bacteria on the basis of structure and composition of their cell wall. Students will be provided with pure cultures of Escherichia coli, a Gram negative bacterium, and Staphylococcus aureus, a Gram positive bacterium, for the Gram stain procedure. They will also select a colony from the exposed plates from Exercise 1. Students will follow the protocol as illustrated in Figures 4 and 5.

USDA-NIFA-HSI Grant PRE 2010-02088

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Figure 4: Bacterial Smear Preparation.

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Figure 5. Gram Staining Procedure.


USDA-NIFA-HSI Grant PRE 2010-02088

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Exercise 4. Demonstration on rapid methods for the detection of pathogens. Several adjuncts to traditional methods and a demonstratio

USDA-NIFA-HSI Grant PRE 2010-02088

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Exercise 4. Demonstration on rapid methods for the detection of pathogens. Several adjuncts to traditional methods and a demonstration of rapid methods will be done. o The demonstration will allow students to establish a comparison between the traditional methods for detection of microorganisms in foods and the use of molecular biology techniques for such detection. Adjuncts to traditional methods that simplify laboratory work: o API 20E- consists of a plastic strip of 20 individual, miniaturized tests tubes, each containing a different reagent used to determine the metabolic capabilities, and, ultimately, the genus and species of enteric bacteria in the family Enterobacteraceae. o Enterotube- System for the rapid identification of Enterobacteriaceae. It is a self-contained, compartmented plastic tube which contains 12 different growth media and an enclosed inoculating needle that permits simultaneous inoculation of the separate media from a single colony and shows results from 15 standard biochemical tests. o Petrifilm- All in one plating system used to culture various microorganisms. Generally comprises a soluble gelling agent, nutrients, and indicators for activity and enumeration. Rapid methods: o BAX for the detection of E. coli O157:H7, Listeria, and Salmonella. o RT-PCR (Real Time-Polymerase Chain Reaction).

USDA-NIFA-HSI Grant PRE 2010-02088

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Referencias 1. http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalytical ManualBAM/default.htm 2. http://www.synbiosis.com/acolyte/ 3. http://www.800ezmicro.com/products/63/152-wasp-2-spiral-plater.html 4. http://www.800ezmicro.com/products/colony-counters/130-protocol.html 5. http://www2.dupont.com/Qualicon/en_US/products/BAX_System/bax_ecoli.html

USDA-NIFA-HSI Grant PRE 2010-02088

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