pathogens_training in microbiological analysis of food as of feb. 6

FOOD AND NUTRITION RESEARCH INSTITUTE DEPARTMENT OF SCIENCE AND TECHNOLOGY

Training Manual Microbiological Analysis of Food

PATHOGENS

This Training Manual was developed by the Food Analytical Service Laboratory (Laboratory Services Group) of FNRI-DOST for the purpose of its training courses. This cannot be reproduced in partial or full without the approval of FNRI.

2 Training in Microbiological Analysis of Food Food Analytical Service Laboratory Food and Nutrition Research Institute Department of Science and Technology

Prepared By:

MICROBIOLOGY UNIT

FOOD ANALYTICAL SERVICE LABORATORY (FASL)

LABORATORY SERVICES GROUP (LSG)

2014


TABLE OF CONTENTS

INTRODUCTION.4

(Course Description, Objectives and Mechanics)

TOPICS

Topic 1. Introduction to Food Microbiology..6

Topic 2. General Laboratory Practices........................10

LABORATORY OBSERVATION....15

METHODS OF ANALYSIS

METHOD1. Detection of Salmonella ......18

METHOD 2. Enumeration of Staphylococcus aureus ....24

METHOD 3. Enumeration of Bacillus cereus .29

METHOD 4. Detection of Listeria monocytogenes...34

ANNEXES

ANNEX A Microbiology Laboratory Safety Guidelines .....40

ANNEX B Media Preparation.41

ANNEX C Biochemical tests..42

ANNEX D Workshops/Sample Worksheets...46

ANNEX E Training Schedule.53


INTRODUCTION

Duration: One (1) hour

Learning Objectives

General: To discuss the course objectives, course content, significance of the

course, schedule of training and expected output.

Specific: After the session, the participants should be able to:

1. enumerate the objectives of the course;

2. appreciate the importance of the course in detection and/or

enumeration of pathogens; and

3. give their expectations on the course and ensure that it will be

included in the training objectives.

Training Method: Lecture with visuals and discussion; Pre-evaluation (short quiz).

Materials Needed: Lecture presentation, blank CD, laptop computer and LCD

projector, blank cassette tapes and cassette recorder for

documentation, office supplies and materials, white board marker

and eraser, quiz papers.

Content: Introduction / Overview of the Course

Course Description:

This training on microbiological analysis of food describes the testing

procedures for detection and/or enumeration of specific pathogens such as

Salmonella, Listeria monocytogenes, Staphylococcus aureus, and Bacillus

cereus. It also includes a brief introduction about food microbiology, updates on

emerging foodborne pathogens and standard methods for their detection

according to regulations. Also, it highlights the preparation and use of quality

assurance program in a food testing laboratory.

Training Objectives:

After the session, the participants should be able to:

(a) explain the importance of microbiological analysis on food samples;


(b) identify the different procedures for laboratory quality assurance and

apply good laboratory practice in the laboratory;

(c) conduct proper sampling, sample preparation and sample storage;

(d) enumerate the laboratory requirements (e.g. equipment, facilities,

reagents, laboratory supplies), and the general principles of the test

methods; and

(e) conduct microbiological analysis to detect specific pathogens such as

Salmonella, Listeria monocytogenes, Bacillus cereus, and

Staphylococcus aureus from a given food sample in the laboratory.

Training Schedule (see attached)

Training Materials

1. LCD Projector

2. Lecture presentation/ materials

3. Blank CD

4. White Board/ White Board Marker/ Eraser

5. Sound System and Microphone (Lecture)

6. Laser pointer

7. Cassette recorder and blank cassette tapes for documentation

8. Office supplies and materials (bond paper, pens, pencils, etc.)

9. Equipment/facilities, reagents and lab. Supplies for the observation

training

10. Laboratory gown

Resource Persons / Trainers FASL staff


Topic 1. INTRODUCTION TO FOOD MICROBIOLOGY

Duration: One (1) hour

Learning Objectives

General: To discuss updates/ knowledge on Food Microbiology


1. discuss Food Microbiology and its importance;

2. discuss food contamination and food safety;

3. define the existing standards and regulatory offices in the

Philippines concerning the microbiological quality of food.

Training Method: Lecture with visuals and discussion.

Materials Needed: Lecture presentation, blank CD, laptop computer and LCD

projector, office supplies and materials, white board marker and

eraser.

Content: Food Microbiology

Definition and Scope of Food Microbiology

Food Microbiology encompasses the study of microorganisms, which have both

beneficial and deleterious effects on the quality, and safety of different food

products. It focuses on the general biology of the microorganisms that are found in

the foods including their growth characteristics, identification, and pathogenesis.

Specifically, food microbiology is mostly interested on areas such as food poisoning,

food preservation, food spoilage, and food legislation.

Basic Microbiology in a glance

Microbiology is the science that deals with the study of microorganisms including

algae, bacteria, fungi, protozoa and viruses. The most abundant of all are bacteria.

Bacteria are unicellular organisms that are relatively small (0.2 to 5 m). They

reproduce asexually and have specific nutritional requirements, temperature,

humidity, pH, etc. While bacteria are most often associated with spoilage and

poisoning, some species help in the food preservation. Meanwhile, foodborne fungi

include the molds and yeasts. Molds are filamentous fungi that grow in the form of

tangled mass spreading rapidly. Yeasts, on the other hand, are unicellular fungi

reproducing by budding.


Parameters that affect growth of microorganisms in foods

The following is a list of parameters that can either result to promotion of

microorganisms growth or their inhibition in certain food products.

pH

Moisture Content (Water activity)

Oxidation-reduction potential

Nutrient content. And antimicrobial constituents

Biological structures

Storage temperature and relative humidity

Presence/absence of gases

Food Contamination

In the food production chain, there are several points by which food can become

contaminated such as from the farm, processors, retailers, and consumers. These

critical points must be considered during sampling and analysis.

Food Safety Concerns:

It is a fact that diseases caused by food borne pathogens create a public health

problem worldwide. Preventing these diseases is another issue. Globally, combined

institutions and sectors of various nations have committed to improve food safety.

Countries have set microbiological criteria for raw or finished processed products.

In the Philippines, the Food and Drug Administration (Department of Health)

issued guidelines for the assessment of microbiological quality of certain processed

foods under the FDA Circular no. 2013-010. The National Meat Inspection Service

(Department of Agriculture) also issued guidelines on the assessment of

microbiological quality of fresh, chilled and frozen meat under Memorandum

Circular 9-2008-05. Several standards specific for different food products are listed

under the Philippine National Standards for foods, which are complied and

regulated by the Bureau of Product Standards (Department of Trade & Industry).

Note: These Circulars and Standards are available online.

Emerging Pathogens

Salmonella sp.

Salmonellosis is one of the most common cause of infectious diseases

transmitted by contaminated poultry foods. A critical goal in food processing

plant and governmental control agencies is to prevent Salmonella

contamination of food products and this prevention depends to a great extent

on an adequate quality control program. Salmonella detection is still highly


dependent on employing appropriate culture media. Appropriate enrichment is

important in reviving cells that may be stressed during processing of the food.

Bacillus cereus

Bacillus cereus are Gram-positive, facultatively anaerobic, endospore forming large rods widespread in the environment and are often isolated from soil and vegetation. It can implicate two types of illness. The first type, called diarrheal type, is caused by consuming contaminated food. Inside the small intestine, the bacterium produces an enterotoxin, a large molecular weight protein, which leads to diarrhea. The other type, called vomiting or emetic type, is associated with a different kind of toxin produced by B. cereus. The emetic toxin produced is a low molecular weight, pH-stable and heat- and protease-resistant toxin that leads to nausea and vomiting. Diarrheal type of food poisoning is often associated with meats, milk, vegetables and fish while the vomiting type outbreaks are implicated with rice products and other starchy products.

Staphylococcus aureus

Staphylococcus aureus are small and spherical (cocci) Gram-positive, catalase positive bacteria appearing in pairs, short chains or in grape-like clusters and are widely distributed in the environment. It is one of the most resistant non-spore forming human pathogen and can survive for extended periods in aa dry state. It is a versatile pathogen, which is the causative agent of Staphylococcal food posioning, toxic shock syndome, pneumonia, post-operative wound infection and nosocomial bacterimia. Importantly, it causes sporadic food poisoning episodes around the world and the foods often implicated include meat and meat products; poultry and egg products; salads; bakery products; and milk and dairy products.

Listeria monocytogenes

Listeria monocytogenes was discovered as a pathogen of animals and

humans in the 1930s. As far as humans are concerned the organism was

initially identified as a cause of abortion in early pregnancy, stillbirth, or of

septicemia after an uneventful birth. Ecological surveys have demonstrated

that Listeria in general, and L. monocytogenes in particular, are naturally

occurring in a wide variety of domestic animals, particularly sheep and

chickens. L. monocytogenes has four attributes: the elevated heat resistance,

the ability for relatively rapid growth at refrigeration temperatures, a marked

tolerance of reduced pH values, and growth in the presence of over 5%

sodium chloride. L. monocytogenes have been isolated from raw staple foods

including chicken,red meat, seafood, and, of course, raw milk

Escherichia coli 0157:H57

Clostridium perfringens

Clostridium botulinum


Campylobacter

Yersinia

References:

Jay, J., M.J. Loesnner & D.A Golden. (2005). Modern Food Microbiology.(7th ed).

USA: Springer Science Business Media.

Lampel, K.A., Al-Khaldi, S. and Cahill, S.M. Bad Bug Book: Handbook of

Foodborne Pathogenic Microorganisms and Natural Toxins. Food and

Drug Administration, USA:2012. 2nd edition.


Topic 2. GENERAL LABORATORY PRACTICES

Duration: One (1) hour lecture and one (1) hour laboratory

Learning Objectives

General: To discuss the guidelines to follow in the laboratory during the

conduct of microbiological analyses of pathogens.


1. explain the significance and principles of laboratory quality assurance;

2. apply good laboratory practice and quality assurance in the laboratory;

and

3. identify the different monitoring procedures involved in a food

processing environment and explain the purpose of each practice.

Identify and understand the quality control procedures

employed in the analysis; and

Implement the quality control procedures in the laboratory.

Training Method: Lecture with visuals, Discussion and Laboratory Demo/Tour.

Materials Needed: Lecture presentation, blank CD, laptop computer and LCD projector,

blank cassette tapes and cassette recorder for documentation, office

supplies and materials, white board marker and eraser.

Content: General Laboratory Practices

Basic Food Microbiology Skills

In testing for pathogens, it is a must that the analyst is familiar and has gained

skills on the following concepts:

1. Materials and Media Preparation (Refer to Annex D: Workshop 1)

2. Food Sampling and Preparation of Food Homogenate

3. Identification and Confirmation of Isolates

Plating skills such as Streaking for Isolation, Pour and spread plating for

enumeration are necessary in this training. Isolation of cultures is an important

step in confirming and identifying if such are the target organisms. Isolated

cultures are used in a series of biochemical and confirmation tests.

4. Gram staining, Spore Staining and Microscopy


Gram staining, which is an initial step in microbial identification, is a useful aide

in differentiating the major groups of bacteria based on their cell wall

composition: the Gram positive and Gram negative bacteria. It also helps in

morphological characterization of isolates.

5. Observation and Interpretation of results

A good analyst is able to observe and properly interpret results. This is gained

through adequate training and experience.

Good Laboratory Practice

Good Laboratory Practice (GLP) describes how microbiologist and other technical

staff conduct analytical work. It includes planning, monitoring, recording, archiving

and reporting results. The goal of GLP is to get the right results. It is a must to

make all necessary preparations:

Before analysis

Make a plan of all activities such as locating the samples, reviewing the

up-to-date copy of the method available, checking that all equipment are

in good working conditions, assuring that all materials, glass ware, and

other supplies are available, sterile and properly dried, if necessary,

checking that the environmental conditions specifications are met,

ensuring that the working area is clean and disinfected, and all other

quality assurance measures are performed and results have passed the

conditions.

During analysis

Note that samples are ready (thawed out properly, if needed). Sampling

and entire analysis must be done aseptically. It is important that the

method is followed exactly as it is written. Observations and/or readings,

colony counts, biochemical reactions should be clearly and directly

recorded on the worksheets. During analysis, reference working cultures

should be used as positive and negative controls. Blank controls must

also be used to ensure sterility of the environment or of the media being

used.

After analysis

After analysis, results must be calculated accurately once all data are

recorded properly. All the laboratory glass ware and supplies used in the

analysis should be washed as soon as possible, decontaminating prior to

washing, if potential microorganisms are present. Hazardous chemicals and

decontaminated media should be disposed properly by rinsing and/or


decontamination. Equipment used must also be cleaned and/or disinfected

carefully after the work has been conducted. Samples analyzed must be

retained for a specific period of time until Report of Analysis has been

released to the customer, or as specified to the laboratorys quality manual.

Reporting the Results

Results should be translated into clear information in such a way that the

customer can understand it. Also, these must be expressed to the correct

number of significant figures or decimal places and mode of expression.

Quality Assurance in the Laboratory

General Laboratory Operations: Components of the QA program

Sample Management

Laboratory Standard Operating Procedures

Personnel

Facilities

Equipment and Instrumentation

Laboratory Glassware

Media and Reagents

Record keeping

General Safety Guidelines

The general safety guidelines being followed and implemented in the Food

Analytical Service Laboratory are written on the appendix A.

Sampling

Obtaining a proper sample for the conduct of analytical testing is of first priority.

A sample is required to be a representative of the entire lot of the food material

being evaluated. The sample must also be of proper type for the determination to

be made or for the requested analytical parameters. Lastly, the sample must be

protected against contamination and improper handling.

An appropriate sampling plan should be obtained and described by the person

acquiring and submitting the samples to the laboratory. This includes procedures

of collecting, labeling, transporting, storing and preparing samples for analysis.

Detailed sampling plans for specific food types are described in literatures.

A proper sample subjected to analysis is very important such that

laboratory results and their interpretation will only be considered valid if


samples submitted to the laboratory are representative of the lot/batch

under study or investigation.

Steps

Sample Collection

In collecting the sample, the physical state 9form, shape, particle size)

of the food product must be considered to ensure that the number of units

will be representative and/or statistically significant for its intended use.

Sample Handling and Storage

Prior to shipment, samples must be stored properly. For frozen

products, store at -200C. For refrigerated products, store at 40C. During

transport, an insulated material must be used. Dry products, on the other

hand, can be packed in a cardboard box-stacked and positioned

appropriately to prevent breakage. If possible, these samples must be

submitted with its original unopened packaging and be delivered to the

laboratory as soon as possible. Also, samples must be labeled properly,

clearly, and completely.

Receipt of Samples

Upon receipt, the general conditions of the sample must be noted.

Personnel receiving the sample must label and record them accurately.

Analysts must be able to analyze them immediately, if possible or if not,

be stored properly and appropriately.

Preparation of Food Homogenate for Analysis

During the preparation of food homogenate from the original sample,

aseptic conditions must be followed. Surrounding working areas must be

cleaned and disinfected as well as the hands of the analysts.

Liquid samples are thoroughly shaken while dry samples are mixed to

obtain an even distribution of microorganisms in the sample. Samples are

weighed accurately and are mixed with the appropriate diluents. Mixing of

the analytical unit to produce a food homogenate can be done by blending

or stomaching. It must be noted that upon obtaining the food homogenate,

all dilutions and inoculation of the sample to the appropriate media must

be done within 15 minutes.

Several food types require specific procedures for sampling. Consult

literatures for such.


The Use of Working Microbial Cultures during analysis

The use of positive control cultures, as well as negative control cultures, is

absolutely necessary to verify that the analysis will detect the target organism.

Microbial Monitoring of Laboratory Environment

Like the food processing environment, the food microbiology laboratory

environment must also be monitored such that sampling sites, which are possible

to harbor microorganisms that may directly or indirectly contaminate the food

product, are selected. This is especially true when working with enriched cultures

or highly contaminated samples. Monitoring is performed to verify the

effectiveness of cleaning and disinfection practices and to determine the presence

of pathogens in the working environment. Several equipment and critical work

surfaces should be included in the routine monitoring. Strategies include Surface

Contact methods and Air sampling methods.

Conclusion

The application of all the principles discussed achieves the following:

Prevention of cross-contamination of samples being examined

Protection of the personnel against infection and protection of the

environment against contamination

Assurance of the correctness of data generated from analyses through

monitoring and verification of QA system

Expression of suitability for use of the analytical procedures

References

Andrews, W.H. & Hammack, T.S. (2003). Chapter 1: Food Sampling and Preparation

of Food Homogenate. Bacteriological Analytical Manual. Retrieved from

http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063335.htm

Downes, F & Ito, K. (2001). Compendium of Methods for Microbiological

Examination of Foods. (4th ed). Washington, DC: American Public Health

Association.


LABORATORY OBSERVATION- MICROBIOLOGICAL ANALYSIS OF

FOOD : Test for Pathogens

Duration: Fifteen (15) hours laboratory

Learning Objectives

General: To discuss the techniques and quality control procedures employed

in the detection and/or enumeration of pathogens such as Salmonella

sp., Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus.


1. Follow the procedures of the test methods, apply good laboratory

practices and conduct microbial analysis in the laboratory

properly.

2. Apply and implement quality control procedures in the

laboratory

Training Method: Observation training in the Food Analytical Service Laboratory on

microbiological analysis.

Method 1 Detection of Salmonella sp.

Method 2 Enumeration of Staphylococcus aureus

Method 3 Enumeration of Bacillus cereus

Method 4 Detection of Listeria monocytogenes

Materials Needed: Laboratory supplies, Culture media/reagents, equipment for

microbiological analysis, notebook, calculator, laboratory gown,

Working Reference Cultures, Worksheets


METHODS

OF

ANALYSIS


CONTENTS

Method 1 Detection of Salmonella sp. (FDA BAM-5, 2007)

Method 2 Enumeration of Staphylococcus aureus (FDA BAM-12, 2001)

Method 3 Enumeration of Bacillus cereus (FDA BAM-14, 2001)

Method 4 - Detection of

Listeria monocytogenes (FDA BAM-10, 2011)


METHOD 1

Detection of

Salmonella sp.

(Culture based method)


Method 1 Detection of Salmonella sp.

1. PURPOSE/SCOPE

This method of analysis is intended to detect the presence of Salmonella sp. in a

given food sample. This involves the enrichment of a portion of the sample, then

inoculation to selective secondary enrichment broths, then streaking to selective

agars. After which, confirmatory tests are performed using classical biochemical

tests.

2. SAFETY and PRECAUTIONS

2.1 Perform method in aseptic conditions, using Biological Safety Cabinet

(Class II).

2.2 Wear proper laboratory attire: laboratory gown, mask, hair cap, gloves,

closed shoes.

2.3 Precautions in media preparation and conventional culture procedure

must be observed and considered for the reliable isolation of the target

organism.

3. REFERENCES

3.1 FDA Bacteriological Analytical Manual, Chapter 5: Salmonella. 2011

3.2 Compendium of Methods for the Microbiological Examination of Foods. 4th

ed.

3.3 Modern Food Microbiology. J. M. Jay. 1996. 5th edition.

4. DEFINITION

Salmonella are small, gram-negative, non-sporing rods which are widely

distributed in nature. They are the most important species responsible for

foodborne gastroenteritis. Generally, they are unable to ferment lactose, sucrose,

or salicin, although glucose and certain other sugars are fermented, with H2S

production.


5. PRINCIPLE

This culture based method is a qualitative test, which determines the presence or

absence of Salmonella in a given sample. It starts with the pre-enrichment step

using a non selective medium to resuscitate Salmonella organisms, which are

usually in low numbers in food. This is followed by selective enrichment step,

using two selective medium, to increase recovery of Salmonella. Then, three

selective agar are used for isolation of colonies. These selective media enable

the distinction of Salmonella from non-Salmonella bacteria. Subsequently,

biochemical screening is performed to characterize isolates. However, complete

identification should not be solely based on the biochemical tests since

Salmonella do not always produce typical biochemical reactions.

6. CULTURE MEDIA AND REAGENTS

All media shall be of recognized of quality. The reagent water used shall be

distilled water. Note: Culture media and reagent water should undergo quality

control check (intermediate) before use.

6.1 Lactose broth

6.2 Bactopeptone Water

6.3 Tetrathionate Broth

6.4 Rappaport-Vassiliadis Broth

6.5 Xylose Lysine Decarboxylase Agar

6.6 Bismuth Sulfite Agar

6.7 Hektoen Enteric Agar

6.8 Triple Sugar Iron Agar

Dispensed in slant positions to tubes

6.9 Lysine Iron Agar

Dispensed in slant positions to tubes. Prepare with a deep butt (4cm).

6.10 Phenol Red Broth with sugar solutions (glucose, sucrose, dulcitol,

lactose)

Prepare the phenol red broth base with 0.5% sugar solutions.

6.11 MacConkey Agar

6.12 Tryptone broth

6.13 Trypticase soy broth/Nutrient broth

6.14 MR-VP Broth

6.15 SIM Medium

6.16 Simmons Citrate Agar

6.17 Urea broth

6.18 Lysine decarboxylase broth


6.19 Kovacs Reagent

6.20 VP Reagents

6.21 Methyl red indicator

6.22 0.85% Physiological saline solution

6.23 API 20E kit

7. EQUIPMENT AND MATERIALS

7.1 Biological Safety Cabinet

7.2 Waterbath, at 43 + 2 0C; for RV incubation

7.3 Stomacher

7.4 Top Loading Balance, calibrated

7.5 Hotplate/Microwave Oven, for melting solidified culture medium

7.6 Incubator, set at 350C

7.7 Sterile pipettes

7.8 Sterile plates

7.9 Sterile tubes

7.10 Sterile wide mouth, screw cap jars/bottles

7.11 Inoculating loop and loop sterilizer

7.12 Autoclave

8. PROCEDURE

8.1 Preparation of Sample Homogenate

8.1.1 Refer to Topic 3: Sampling

8.1.2 Measure 25 analytical unit from the food sample aseptically.

8.2 Pre - enrichment

8.2.1 Mix the 25 g or ml of food sample with 225 ml of Lactose broth (or

the appropriate diluent) in a sterile container.

8.2.2 Incubate for 18-24 hours at 350C.

8.3 Selective enrichment

8.3.1 Transfer 1.0ml of the incubated homogenate to 10mL freshly

prepared Tetrathionate broth. Incubate for 24 hours at 35 0C.


8.3.2 Inoculate also 0.1ml of the homogenate to 10mL RV broth. Incubate

at 430C waterbath for 24 hours.

8. 4 Inoculation to selective media

8.4.1 After incubation, streak for isolation a loopful of inoculum from TT

broth to each selective medium: XLDA, BSA, and HEA.

8.4.2 Do the same for each loopful from RV broth.

8.4.3 Invert the plates and incubate at 350C for 18-24 hours.

8.5 Reading of plates

8.5.1 After incubation, observe plates for growth of typical Salmonella.

XLDA: colorless colonies with or without black center

BSA:

HEA: blue to blue green colonies with or without black center

8.5.2 If plates have mixed cultures, re-streak to MacConkey Agar to get a

pure isolate.

9. CONFIRMATION OF RESULTS

9.1 Pick at least 2 colonies from each selective agar.

9.2 Inoculate each isolate to TSI (streak then stab) and LIA (double stab then

streak) tubes. Incubate for 24 hours at 350C.

9.3 Observe for red (alkaline) slant and yellow (acid) butt in TSI; purple

(alkaline) reaction in LIA butt of tube.

9.4 Subject the presumptive TSI cultures to urease test.

9.5 Test urease-negative cultures with the following biochemical tests: lysine

decarboxylase test and dulcitol fermentation test.


9.6 From the TSI culture, inoculate small growth to tryptone broth and perform

KCN, Malonate and Indole test.

9.7 When isolates are not conclusive of Salmonella, additional biochemical

tests may be performed such as lactose and sucrose fermentation tests,

MR-VP and citrate test.

9.8 As an alternative, available commercial kits can be used for rapid

identification of Salmonella.

9 REPORTING OF RESULTS

Report as Positive or Negative for Salmonella per 25 g of the given food

sample.


METHOD 2

Enumeration of


(Direct Plate Count

method)


Method 2 Enumeration of Staphylococcus aureus

1. PURPOSE/SCOPE

1.1 This method of analysis is intended to enumerate the coagulase-positive

Staphylococcus aureus in food samples. Since Staphylococcus aureus is

highly vulnerable to destruction by heat treatment and many sanitizing

agents. Foods must be examined for S.aureus to confirm if the organism

is the causative agent of foodborne illness, to determine whether a

food/food ingredient is a potential source of enterotoxigenic staphylococci,

and to demonstrate post-processing contamination.



(Class II).


closed shoes.

3. REFERENCES

3.1 FDA Bacteriological Analytical Manual, Chapter 10: Staphylococcus

aureus. 2001.


ed.

4. DEFINITION

Staphylococcus aureus are gram positive cocci. Their presence indicates

potential public health hazard since many strains produce enterotoxins causing

food poisoning if ingested.

5. PRINCIPLE

This direct plate count method is suitable for the analysis of foods in which more

than 100 S.aureus cells per gram may be expected.






6.1 Baird Parker Agar

Prepare as described in the Manufacturers Instructions. Separately

prepare the agar base in the appropriate amount and sterilize at 1210C for

15 minutes. Cool the agar at 450C through circulating water bath and

aseptically add the egg yolk tellurite emulsion in proportion. Mix and

dispense about 18 to 20 ml in Petri dishes. Dry the plates prior to use.


Dissolve 1 gram of BPW in 1.0 L distilled water. Dispense 225 ml and 90

ml to dilution bottles. Sterilize by moist heat at 1210C for 15 minutes.

6.3 Brain heart infusion broth

6.4 Trypticase soy agar

6.5 Coagulase plasma (rabbit) with EDTA

6.6 Hydrogen peroxide, 3% for catalase test

6.7 Phenol red broth base with agar, or any carbohydrate fermentation

medium; with Glucose and Mannitol sugar solutions

Prepare 0.5% filter sterilized sugar solutions and aseptically add to phenol

red agar (sterilized and dispensed in tubes).



7.2 Waterbath

7.3 Stomacher





7.8 Sterile plates

7.9 Sterile tubes




7.12 Autoclave

8. PROCEDURE



8.1.2 Mix the 25 g or mL food sample to 225 mL Buffered peptone water

and stomached for 30 seconds.

8.2 Dilution and Inoculation

8.2.1 Prepare decimal dilutions and shake dilutions vigorously for 25

times.

8.2.2 Inoculate 1mL sample suspension to 3 BPA plates distributing

inoculum equitably (0.3. 0.3, 0.4 mL).

8.2.3 Spread inoculum over surface of agar plate using sterile bent glass

rod. Retain plates in upright position until inoculum is absorbed by

the agar.

8.2.4 Invert plates and incubate for 44-48 hours at 350C.

8.3 Counting and Recording Colonies

8.3.1 Observe for typical appearance of S. aureus which is circular,

smooth convex, moist, 2-3mm in diameter, gray to jet-black with off

white margin, surrounded by opaque zone, and with an outer clear

zone.

When touched with inoculating needle, colonies have buttery or

gummy consistency.

8.3.2 Select plates with 20-200 colonies. If plates have different types of

colonies, count colonies for each type, and record separately.

8. 4 Coagulase test


8.4.1 Transfer suspect S. aureus colonies into small tubes with 0.2-0.3 ml

BHI broth and emulsify thoroughly.

8.4.2 Inoculate TSA slant with the BHI suspension, for ancillary tests.

Retain these slant cultures at room temperature.

8.4.3 Incubate BHI tubes at 350C for 18-24 hours.

8.4.4 Add 0.5 ml reconstituted coagulase plasma with EDTA to the BHI

culture and mix thoroughly. Incubate at 350C and examine

periodically for a total of 6 hours. Observe for clotting.

8.4.5 Only firm and complete clotting is considered as positive for S.

aureus.

8.5 Ancillary tests

8.5.1 If partial clotting is observed, further testing is a must.

8.5.2 Using the growth from the TSA slant, perform Gram staining (for

microscopic observation), catalase test, anaerobic utilization of

glucose and mannitol, lysostaphin sensitivity, and thermostable

nuclease production.

8.6 Computing of results

8.6.1 Add number of colonies on triplicate plates represented by colonies

giving positive coagulase test and multiply by dilution factor. Use the

formula below for computation.

=

9. REPORTING RESULTS

Report as CFU per gram or ml of Staphylococccus aureus


METHOD 3

Enumeration of

Bacillus cereus

(Direct Plate Count

method)


Method 3 Enumeration of Bacillus cereus

1. PURPOSE/SCOPE

This method of analysis is intended to enumerate Bacillus cereus. This

organism causes food poisoning when foods are prepared and held without

adequate refrigeration for several hours before serving. B.cereus is widely

distributed in nature and can be isolated from a variety of foods.



(Class II).


closed shoes.

3. REFERENCES

3.1 FDA Bacteriological Analytical Manual, Chapter 3: Aerobic Plate Count.

2001.


ed.

4. DEFINITION

Bacillus cereus is an aerobic spore forming bacterium commonly found in soil,

on vegetables, and in many raw and processed foods.

5. PRINCIPLE

Plate count method for enumerating Bacillus cereus in food samples make use of

MYP culture medium, which produces colonies of the target organism

surrounded by precipitate zone. This indicates lecithinase production. B.cereus,

in most instances, produces very strong reaction in egg yolk agar, which is

characterized by a wide zone of turbidity surrounding the individual colonies after

20-24 hour incubation.






6.1 Mannitol yolk polymyxin agar or Bacara agar

Prepare as described in the Manufacturers Instructions. Prepare

agar base separately by dissolving dehydrated medium to the appropriate

volume of water. Sterilize at 1210C for 15 minutes. Cool agar at 450C

through circulating water bath and aseptically add 50% egg yolk emulsion

and the Polymyxin B solution in appropriate amounts.


Dissolve 1 gram of BPW in 1.0 L distilled water. Dispense 225 ml and 90

ml to dilution bottles. Sterilize by moist heat at 1210C for 15 minutes.

6.3 50% egg yolk emulsion

6.4 Polymyxin B solutions

6.5 Phenol red glucose broth

6.6 Tyrosine agar

Prepare Nutrient agar and sterilize at 1210C for 15 minutes.

Prepare also Tyrosine solution and sterilize at same conditions.

Aseptically add tyrosine to the nutrient agar and dispense in tubes (and

dry it in slants).

6.7 Trypticase soy-sheep blood agar

Prepare trypticase soy agar and sterilize at 1210C for 15 minutes. After

sterilization and cooling, aseptically add 5ml of defibrinated sheep blood

to 100 ml agar. Dispense in plates.

6.8 Motility medium

6.9 Nitrate broth, and nitrite detection reagents

6.10 Lysozyme broth

6.11 Modified VP medium, and VP Reagents

6.12 Gram stain kit



7.2 Waterbath

7.3 Stomacher






7.8 Sterile plates

7.9 Sterile tubes



7.12 Autoclave

8. PROCEDURE



8.1.2 Mix the 25 g or mL food sample to 225 mL Buffered peptone water

and stomached for 30 seconds.

8.2 Dilution and Inoculation

8.2.1 Prepare decimal dilutions and shake dilutions vigorously for 25

times.

8.2.2 Inoculate 0.1mL sample suspension to properly dried duplicate MYP

agar plates with each dilution of sample.

8.2.3 Spread inoculum over surface of agar plate using sterile bent glass

rod. Retain plates in upright position until inoculum is absorbed by

the agar.

8.2.4 Invert plates and incubate for 18-24 hours at 300C.

8.3 Counting and Recording Colonies

8.3.1 Observe for pink colonies surrounded by precipitate zone (indicating

lecithinase production). Color becomes intense after additional

incubation.

8.3.2 Select plates with 15-150 colonies. Pick at least 5 presumptive

B.cereus colonies and transfer to Nutrient agar slants. Incubate

slants 24 hours at 300C


8. 4 Confirmatory tests

8.4.1 Gram stain

Prepare gram-stained smears from slants and examine

microscopically. B.cereus appear as large Gram positive bacilli in

short to long chains; with ellipsoidal spores, centrally to

subterminally located; do not swell sporangium.

8.4.2 Other tests

Transfer 3mm loopful of culture from each slant to a sterile tube

containing phosphate buffered dilution water and suspend it. Use

the suspended culture to inoculate on the ff media for confirmatory

tests: Phenol red glucose broth, tyrosine agar, nitrate broth, modified

VP medium, and lysozyme broth.

8.6 Computing of results

8.6.1 Calculate number of Bacillus cereus cells per gram or ml of sample

using the formula below:

= . .

9. REPORTING RESULTS

Report as CFU per gram or ml of Staphylococccus aureus


METHOD 4

Detection of


(Culture based method)


Method 4 Detection of Listeria monocytogenes

1. PURPOSE/SCOPE

This method of analysis is intended to detect the presence of Listeria

monocytogenes in a given food sample. It allows the analysis of foods that

contain injured L.monocytogenes cells and high populations of contaminants.

The target organism is known to be universally occurring in food and is a

well known hazard causing listeriosis, which often leads to severe

consequences, particularly in susceptible subpopulations.


Note: The organism to be handled is highly virulent in nature so, strict

safety precautions are demanded when working.


(Class II).


closed shoes.

2.3 Be especially mindful of generating aerosols during blending and mixing

procedures and be meticulous in rinsing work areas often with

bactericidal solutions.

2.4 Pregnant women or other immune-compromised personnel are prohibited

from entering laboratories in which L.monocytogenes will be analyzed.

3. REFERENCES

3.1 FDA Bacteriological Analytical Manual, Chapter 10: Detection and

Enumeration of Listeria monocytogenes. 2011


ed.

4. DEFINITION

Listeria monocytogenes is a short, gram-positive, nonsporeforming rod-shaped

bacterium that appears coccoidal in older cultures. It thrives under anaerobic to


microaerophilic conditions, preferring 10% carbon dioxide environment. It grows

over a pH range of 5 to 9.6.

5. PRINCIPLE

Successful isolation of Listeria monocytogenes depends on the choice of

method sensitive to the recovery of low-level contamination. This method

employs pre-enrichment media incubated at 350C followed by plating on

selective/differential agars. Numerous taxonomic tests are required for

confirmation. This method also utilizes the distinct ability of the organism to

display resistance to many antibiotics, which are incorporated at the pre-

enrichment medium.





6.1 Buffered Listeria Enrichment broth

6.2 Selective supplements for BLEB

Prepare the following supplements as stock solutions and filter-sterilize

them: Acriflavin HCl (10mg/L), Nalidixic acid, Sodium salt (40mg/L),

Cycloheximide (50mg/L). Store at 40C and protect from light. Aseptically

add to enrichment after 4 hr incubation (Refer to procedures below).

6.3 Oxford Medium

6.4 CHROMagar Listeria

6.5 3% Hydrogen peroxide, for catalase test

6.6 Sheep blood agar

Prepare Blood base no.2 as recommended and sterilize at 1210C for 15

minutes. Cool to 500C and aseptically add 5% defibrinated sheep blood.

Dispense in plates.

6.7 Gram stain kit

6.8 Carbohydrate fermentation medium, with sugar stock solutions

Prepare carbohydrate fermentation medium (Purple carbohydrate

fermentation broth base), dispense 2.5 ml into tubes with fermentation

tubes and sterilize at 1180C for 10 minutes.

Prepare 0.5% solutions of dextrose, esculin, maltose, rhamnose,

mannitol and xylose. Filter sterilize each solution.

Aseptically add each solution to a tube.


6.9 Nitrate broth, and Nitrite detection reagents

6.10 Trypticase soy agar with 0.6% yeast extract (TSAYE)

6.11 Trypticase soy broth with 0.6% yeast extract (TSBYE)

6.12 SIM Medium

6.13 API Listeria



7.2 Waterbath

7.3 Stomacher



7.6 Incubators, set at 300C and 350C


7.8 Sterile plates

7.9 Sterile tubes



7.12 Autoclave

8. PROCEDURE


Refer to Topic 3: Sampling

8.2 Enrichment

8.2.1 Mix the 25 g of food sample with 225 ml of Buffered Listeria

enrichment broth in a sterile container.

8.2.2 Incubate for 4 hours at 350C.

8.2.3 Aseptically add the selective supplements after 4 hours incubation

and continue incubation up to 48 hours.

8.3 Inoculation

8.3.1 Streak a loopful of the incubated homogenate to OXA agar and also

to CHROMagar Listeria, as an option.


8.3.2 Incubate plates at 350C for 24-48 hours.

8.3.3 Observe for black colonies with black halo due to esculin hydrolysis

in OXA medium. For CHROMagar, observe Listeria monocytogenes

for blue to blue-green colonies.

8. 4 Isolation

8.4.1 Pick at least 5 isolates and streak for isolation to TSAYE plates.

8.4.2 Incubate TSAYE plates at 300C (if motility will be observed by wet

mount) or at 350C.

8.4.3 Use these cultures for confirmatory tests. Prepare also Gram-

stained smears from 16-24 hr cultures. Listeria are short gram

positive rods.

9. CONFIRMATION OF RESULTS

9.1 Using the TSAYE cultures, perform the ff tests: catalase test and motility

test. Listeria spp. are catalase positive. In motility test using wet mount,

Listeria spp. can be observed as short, slim rods with tumbling motility. On

the other hand, if SIM is used, umbrella-like growth pattern will be observed

at 7 days room temperature incubation.

9.2 Perform hemolysis test. Listeria monocytogenes produces a slightly

cleared zone around the stab, indicating -hemolysis. If results are

questionable, perform CAMP test.

9.3 From the TSAYE culture, transfer a loopful to TSBYE and incubate at 350C

for 24 hrs. Use this to inoculate carbohydrate fermentation media.

Positively reacting Listeria monocytogenes should produce acid but no gas

in dextrose, esculin, rhamnose and maltose; and must be negative for

mannitol and xylose.

9.4 As an alternative, pure isolates can already be used directly for API Listeria

for identification.

10 REPORTING RESULTS

Report as Positive or Negative for Listeria monocytogenes per 25 g of the

given food sample.


ANNEX

A Microbiology Laboratory Safety

Regulations

B Media Preparation

C Biochemical Tests

D Workshops

E Training Schedule


ANNEX A

MICROBIOLOGY LABORATORY SAFETY REGULATIONS

1. Make a plan of all the activities and make all necessary preparations before

conducting analysis or laboratory work. Read the Standard Operating

Procedures, if not yet familiar.

2. Observe safety precautions at all times.

3. Wear protective clothing (laboratory gown) when entering the laboratory. Also,

Do not wear lab clothing outside the lab

4. Do not eat, drink, chew, or smoke anything in the lab

5. Never, never, never mouth pipette

6. Keep your hands away from your face

7. Always wash your hands before and after analysis (especially after working with

the cultures).

8. Clean and sanitize all working areas by wiping them down with an appropriate

disinfectant (70% alcohol) before and after analysis.

9. Keep the laboratory equipment inside the microbiology lab. When using

instruments and equipment, read and follow the equipment operations manual.

10. Decontaminate used bottles, Petri dishes, test tubes, flasks, and other materials

that may contain potential microorganisms before washing and/or disposal.

Dispose wastes, chemicals and decontaminated cultures/materials properly and

safely.

11. In cases of spills, use forceps and cotton for wiping. Disinfect the affected areas.

Discard cotton/residues into an autoclavable bag. Flame the forceps.

12. Always work with at least another person nearby. Never work alone in the

laboratory.

13. Implement good housekeeping practices to reduce chance of accidents.

14. Follow

15. Label samples, cultures, reagents, and media with permanent markers.

16. Use proper transport vessels (test tube racks) for moving cultures in the

laboratory, and store vessels containing cultures in a leak-proof container when

work with them is complete. Laso, use safety carriers for transporting large

containers of chemicals.

17. Notify safety officer (or supervisor) of all spills, unsafe practices, and accidents.

18. If you do not understand or you are in doubt, PLEASE ASK.

Reference: Food Microbiology: The Laboratory (by Phyllis Entis)


ANNEX B

MEDIA PREPARATION

Basic Steps in Media Preparation;

1. Weigh carefully the proper amount of dehydrated medium

2. Place the requisite amount of distilled water into a suitable container

3. Add the weighed dehydrated medium to part of the water. Mix

4. Add the remaining water and mix again.

5. Check pH and adjust if necessary

6. Heat to boiling to complete dissolution using microwave, hot plate or water bath.

7. Stir often to prevent overheating and burning

8. Distribute medium to appropriate containers, making sure that the amount of

medium per container is no more than 2/3 of the containing volume of the

container.

9. Sterilize at 1210C for 15 minutes or according to the recommended procedures of

the medium.

10. Melt and hold media at 44 to 460C until ready to use, but not exceeding 3 hours.

Reference:

Standard Methods for the Examination of Dairy Products

American Public Health Association

Chapter 4: Media and Dilution Water Preparation/


ANNEX C

BIOCHEMICAL and OTHER CONFIRMATORY TESTS

Anaerobic utilization of Glucose and Mannitol Inoculate tube of carbohydrate fermentation medium containing glucose and mannitol(0.5%). Immediately inoculate each tube heavily with wire loop. Make certain inoculum reaches bottom of tube. Cover surface of agar with layer of sterile paraffin oil at least 25 mm thick. Incubate 5 days at 37C. Run controls simultaneously (positive and negative cultures and medium controls). Acid is produced anaerobically if indicator changes to yellow throughout tube, indicating presence of S. aureus. S. aureus is usually positive in mannitol but some strains are negative. CAMP Test

Streak weakly -hemolytic S. aureus and R. equi vertically on sheep blood agar. Separate vertical streaks so that test strains may be streaked horizontally between them without quite touching them. After 24- and 48-h incubation at 35 C, examine plates for hemolysis in the zone of influence of the vertical streaks.

Hemolysis of L. monocytogenes is enhanced near the S. aureus streak; Catalase Test Use growth from TSA slant for catalase test on glass slide or spot plate, and illuminate properly to observe production of gas bubbles. Citrate Test Inoculate this agar, using needle containing growth from unclassified TSI agar slant. Inoculate by streaking slant and stabbing butt. Incubate 96 2 h at 35C. Read results as follows:

Positive--presence of growth, usually accompanied by color change from green to blue. Negative--no growth or very little growth and no color change.

Most cultures of Salmonella are citrate-positive.

Hemolysis test Inoculate heavily (from TSAye colony) 5% sheep blood agar by stabbing plates that have been poured thick and dried well (check for moisture before using). Draw grid of 20-25 spaces on plate bottom. Stab one culture per grid space. Always stab positive controls and negative control. Incubate for 24-48 h at 35 C. Attempt to stab as near to bottom of agar layer as possible, without actually touching bottom of agar layer and possibly fracturing the agar.

Indole Test Transfer 5 ml of 24 h tryptophane broth culture to empty test tube. Add 0.2-0.3 ml Kovacs' reagent. Record intermediate shades of orange and pink as . Most Salmonella cultures give negative test (lack of deep red color at surface of broth). KCN Test


Transfer 3 mm loopful of 24 h tryptophane broth culture to KCN broth. Heat rim of tube so that good seal is formed when tube is stoppered with wax-coated cork. Incubate 48 2 h at 35C but examine after 24 h. Interpret growth (indicated by turbidity) as positive. Most Salmonella species do not grow in this medium, as indicated by lack of turbidity. Lysine Decarboxylase Test Inoculate broth with small amount of growth from TSI slant suspicious for Salmonella . Replace cap tightly and incubate 48 2 h at 35C but examine at 24 h intervals. Negative test is indicated by yellow color throughout medium. If medium appears discolored (neither purple nor yellow) add a few drops of 0.2% bromcresol purple dye and re-read tube reactions. Salmonella species cause alkaline reaction indicated by purple color throughout medium. Lysostaphin Sensitivity Transfer isolated colony from agar plate with inoculating loop to 0.2 ml phosphate-saline buffer, and emulsify. Transfer half of suspended cells to another tube (13 x 100 mm) and mix with 0.1 ml phosphate-saline buffer as control. Add 0.1 ml lysostaphin (dissolved in 0.02 M phosphate-saline buffer containing 1% NaCl) to original tube for concentration of 25 g lysostaphin/ml. Incubate both tubes at 35C for not more than 2 h. If turbidity clears in test mixture, test is considered positive. If clearing has not occurred in

2 h, test is negative. S. aureus is generally positive. Lysozyme Sensitivity Inoculate 2.5 ml of nutrient broth containing 0.001% lysozyme with 2 mm loopful of culture. Also inoculate 2.5 ml of plain nutrient broth as positive control. Incubate tubes 24 h at 35C. Examine for growth in lysozyme broth and in nutrient broth control. Incubate negative tubes for additional 24 h before discarding. Bacillus cereus grow in the presence of 0.001% lysozyme Malonate Test Transfer 3 mm loopful of 24 h tryptone broth culture to malonate broth. Incubate 48 2 h at 35C, but examine after 24 h. Most Salmonella species cultures give negative test (green or unchanged color) in this broth. Motility Test for B.cereus Inoculate BC motility medium by stabbing down the center with 3 mm loopful of 24 h culture suspension. Incubate tubes 18-24 h at 30C and examine for type of growth along stab line. Motile organisms produce diffuse growth out into the medium away from the stab. Non-motile organisms produce growth only in and along stab. Most strains of B. cereus are motile by means of peritrichous flagella. A few B. cereus strains are also non-motile. Motility Test for L.monocytogenes Inoculate SIM or MTM from TSBye. Incubate for 7 days at room temperature. Observe daily.


Listeria spp. are motile, giving a typical umbrella-like growth pattern. MR-VP test Inoculate medium with small amount of growth from each unclassified TSI slant suspected to contain Salmonella. Incubate 48 2 h at 35C.

1) Perform Voges-Proskauer (VP) test at room temperature as follows: Transfer 1 ml 48 h culture to

test tube and incubate remainder of MR-VP broth an additional 48 h at 35C. Add 0.6 ml -naphthol and shake well. Add 0.2 ml 40% KOH solution and shake. To intensify and speed reaction, add a few crystals of creatine. Read results after 4 h; development of pink-to-ruby red color throughout medium is positive test. Most cultures of Salmonella are VP-negative, indicated by absence of development of pink-to-red color throughout broth.

2) Perform methyl red test as follows: To 5 ml of 96 h MR-VP broth, add 5-6 drops of methyl red

indicator. Read results immediately. A distinct yellow color is negative test.

Most Salmonella cultures give positive test, indicated by diffuse red color in medium. Modified VP Test Inoculate 5 ml medium with 3 mm loopful of culture and incubate tubes 48 2 h at 35C. Test for production of acetylmethyl-carbinol by pipetting 1 ml culture into 16 125 mm test tube and adding 0.6

ml alpha-naphthol solution and 0.2 ml 40% potassium hydroxide. Shake, and add a few crystals of creatine. Observe results after holding for 1 h at room temperature. Test is positive if pink or violet color develops. Bacillus cereus shows positive VP reaction. Nitrate Test Inoculate 5 ml broth with 3 mm loopful of culture. Incubate tubes 24 h at 35C. To test for nitrite, add 0.25 ml each of nitrite test reagents A and C to each culture. An orange color, which develops within 10 min, indicates that nitrate has been reduced to nitrite. Bacillus cereus reduce nitrate to nitrite. On the other hand, Listeria monocytogenes cannot reduce nitrates to nitrite. Phenol Red Glucose test Inoculate 3 mL broth with 2 mm loopful of culture. Incubate tubes anaerobically 24 h at 35C in GasPak anaerobic jar. Shake tubes vigorously and observe for growth as indicated by increased turbidity and color change from red to yellow, which indicates that acid has been produced anaerobically from glucose. A partial color change from red to orange/yellow may occur, even in uninoculated control tubes, due to a

pH reduction upon exposure of media to CO2 formed in GasPak anaerobic jars. Bacillus cereus grow and produce acid from glucose anaerobically. Sugar Fermentation for L.monocytogenes From TSBye culture, inoculate the following carbohydrates as 0.5% solutions in purple carbohydrate broth (the use of Durham tubes is optional): dextrose, esculin, maltose, rhamnose, mannitol, and xylose. Incubate 7 days at 35 C. Positively reacting Listeria spp. produce acid with no gas.


Listeria monocytogenes should be positive for dextrose, esculin, rhamnose, and maltose but negative for mannitol and xylose. Sugar Fermentation Tests for Salmonella spp. Inoculate broth with small amount of growth from TSI culture. Replace cap loosely and incubate 48 2 h at 35C, but examine after 24 h. Production of acid should be interpreted as a positive reaction. Negative test is indicated by no gas formation in inner fermentation vial and red (with phenol red as indicator) or purple (with bromcresol purple as indicator) color throughout medium. Most Salmonella species give positive tests in dulcitol but negative in lactose and sucrose. Thermostable nuclease production Prepare microslides by spreading 3 ml toluidine blue-deoxyribonucleic acid agar on the surface of each microscope slide. When agar has solidified, cut 2 mm diameter wells (10-12 per slide) in agar and remove agar plug by aspiration. Add about 0.01 ml of heated sample (15 min in boiling water bath) of broth cultures used for coagulase test to well on prepared slide. Incubate slides in moist chamber 4 h at 35C. Development of bright pink halo extending at least 1 mm from periphery of well indicates a positive reaction for Bacillus cereus. Tyrosine decomposition Inoculate entire surface of tyrosine agar slant with 3 mm loopful of culture. Incubate slants 48 h at 35C. Observe for clearing of medium near growth, which indicates that tyrosine has been decomposed. Examine negative slants for obvious signs of growth, and incubate for a total of 7 days before considering as negative. Bacillus cereus decomposes L-tyrosine. Urease test With sterile needle, inoculate growth from each presumed-positive TSI slant culture into tubes of urea broth. Include control tubes (uninoculated). Incubate 24 2 h at 35C. Urea broth turn purple-red as a positive test result. Salmonella species are negative for this test, showing no color change in the broth. Wet Mount Motility Pick typical colony from culture plate incubated at 30C or less and examine by wet mount, using 0.85% saline for suspending medium and oil immersion objective of phase-contrast microscope. Choose a colony with enough growth to make a fairly heavy suspension; emulsify thoroughly. If too little growth is used, the few cells present will stick to the glass slide and appear non-motile.

Listeria spp. are slim, short rods with slight rotating or tumbling motility.

Reference:

Bacteriological Analytical Manual Online

(www.fda.gov/Food/FoodScienceResearch/Laboratory/Methods/BacteriologicalAnalyticalManualBAM.htm)


ANNEX D.1

WORKSHOP 1 : MEDIA PREPARATION

SAMPLE MEDIA PREPARATION LOGSHEET

Test for Pathogen

Name of Medium

Brand Lot

Number

Amount weighed

(g)

Volume of water

used (ml)

pH

Sterility Control

Desired incubation

Temp

Room Temp

+ -

Diluent BPW

Bacillus cereus


Salmonella


pH of distilled water used: ______________

Prepared by: ___________________

Date: _________________________


ANNEX D.2

WORKSHOP 2 : ENVIRONMENT MONITORING

SAMPLE ENVIRONMENT MONITORING LOGSHEET

Table 1. Monitoring of Microbiology Laboratory

DAY Date Relative

Humidity Temperature Recorder Remarks

1

2

3

4

5

Table 2. Air Environment Sampling in the Microbiology Laboratory using Open Plate Method

Sampling Sites Media Used Incubation

(Time and Temp.) Results

(CFU per plate) Remarks

(PASS/FAIL) Checked by

WORKSHOP 3 : BASIC MICROBIOLOGY SKILLS

ACTIVITY WORKSHEET

Activity 1 Gram Staining

Gram Staining is a basic procedure necessary to the physical characterization of bacteria. It aims

to differentiate organisms based on their cell wall structure. Gram positive bacteria possess thick

peptidoglycan layer and appear as blue to purple. Meanwhile, Gram negative bacteria have thin

peptidoglycan layer and appear pink to red.

Table 1. Protocol of Gram Staining (Gephardt et al, 1981, Feedback from ASMCUE participants, ASMCUE , 2005)

Reagents Time Purpose

Primary Stain 1 minute

Mordant 1 minute

Decolorizing Agent 15 seconds

Counterstain 30 sec- 1 minute


Activity 2 Spore Staining

Some bacteria such as Bacillus cereus possess primary structures that could not easily be stained

using the typical staining reagents and protocols. Endospore is an example of a structure possessed by

B.cereus. Detection of endospores aid in proper identification and differentiation of several microbial

groups.

Spore staining is a procedure that differentiates vegetative cells from the bacterial endospores.

In this activity, Schaeffer-Fulton method will be used.

Table 2. Protocol of Endospore Staining (Schaeffer-Fulton Method)

Reagents Procedure Purpose

Primary Stain Steam for 5 minutes

Decolorizing agent Wash

Counter stain 30 seconds

Activity 3 Microscopy

Figure 1. Organism: _______________ Figure 2. Organism: ________________

Gram reaction: ___________ Gram reaction: ____________

Figure 3. Organism: __________________


ANNEX D.3

WORKSHOP 4 : ENUMERATION OF Bacillus cereus SAMPLE WORKSHEET

Sample Name: _____________________

Sample Code: ______________________

Bacillus cereus CFU/gram or ml: ____________

Computation:

Analyzed by: ___________________ Checked by: ___________________ Date: _____________

Date received: Date analysis started: Date analysis finished:

___________________ ___________________ ___________________

Dilution

MYPA

GRAM STAIN

SPORE STAIN

Confirmatory tests

A B Colony

Characteristics

Glu

cose

(An

aero

bic

)

Tyro

sin

e

Nit

rate

VP

Lyso

zym

e

Hem

oly

sis

(+)

B.c

ereu

s

100

10-1

10-2

10-3

10-4

CONTROLS

Blank (+) (-)


ANNEX D.4

WORKSHOP 5: ENUMERATION OF Staphylococcus aureus SAMPLE WORKSHEET

Sample Name: _____________________

Sample Code: ______________________

Staphylococcus aureus CFU/gram or ml: ____________

Computation:

Analyzed by: ___________________ Checked by: ________________ Date: _____________


___________________ ___________________ ___________________

Dilution

BPA

COAGULASE TEST

GRAM STAIN

CATALASE TEST

Ancillary tests

Counts Colony

characteristics

Glu

cose

(An

aero

bic

)

Man

nit

ol

(An

aero

bic

)

Lyso

stap

hin

Ther

mo

nu

clea

se

100

0.3

0.3

0.4

10-1

0.3

0.3

0.4

10-2

0.3 0.3 0.4

10-3

0.3 0.3 0.4

10-4

0.3 0.3 0.4

CONTROLS Blank (+) (-)


ANNEX D.5

WORKSHOP 6: DETECTION OF PATHOGENS Salmonella spp.

SAMPLE WORKSHEET

Sample Name: _____________________

Sample Code: ______________________

Salmonella spp. per 25 gram or ml: ____________



___________________ ___________________ ___________________

Colony

characteristics

MAC Urease

Test

Biochemical tests

Salm

on

ella

sp

p

TSI LIA Ly

sin

e

Dec

arb

oxy

lase

DU

lcit

ol KC

N

Mal

on

ate

Ind

ole

Lact

ose

Sucr

ose

MR

VP

Cit

rate

BSA

TT

RV

XLDA

TT

RV

HEA

TT

RV



ANNEX D.6

WORKSHOP 6: DETECTION OF PATHOGENS Listeria monocytogenes

SAMPLE WORKSHEET

Sample Name: _____________________

Sample Code: ______________________

Listeria monocytogenes per 25 gram or ml: ____________



___________________ ___________________ ___________________

Colony

characteristics GRAM STAIN

Catalase test

Motility test

Hem

oly

sis

CM

AP

te

st

Carbohydrate utilization

List

eria

m

on

ocy

tog

enes

Dex

tro

se

Escu

lin

Mal

tose

Rh

amn

ose

Man

nit

ol

Xyl

ose

OXA

CHROM agar



ANNEX E

TRAINING SCHEDULE

Schedule Content Staff Involved

DAY 1

8:00-8:30 Welcome ceremony : Introduction to speakers and participants CSM

8:30-9:00 Course objectives/mechanics/schedule Pre evaluation

9:00-10:00 TOPIC 1: Introduction to Food Microbiology CETC

10:00-10:15 Health Break

10:15-11:15 TOPIC 2: General Laboratory Practices CETC

11:15-11:30 Lab tour

11:30-12:00 Sample Preparation CETC

12:00-1:00 Lunch break

1:00-3:00 Workshop 1: Media Preparation Workshop 2: Environment Monitoring (Open Plate & Use of Swab Technique in Sampling)

CETC/CSM


3:15-4:30 Workshop 3: Gram & Spore Staining & Microscopy

CETC/CSM

DAY 2

8:30-9:30 PRE-LAB: Isolation and detection of pathogens: Salmonella spp. and Listeria monocytogenes

CSM

9:30-10:30 Laboratory Proper: Enrichment and Selective Enrichment CETC/CSM

10:30-10:45 Health break

10:45-12:00 Laboratory Continuation CETC/CSM

12:00-1:00 Lunch Break

1:00-2:00 Streak plating for Isolation CSM

2:00-3:15 Biochemical and Confirmatory tests for Salmonella CSM


3:30-4:30 Confirmatory tests for Listeria monocytogenes CSM

DAY 3

8:30-9:00 PRE-LAB: Enumeration of Staphylococcus aureus CETC

9:00-10:30 Laboratory Proper Enumeration of Staphylococcus aureus

CETC


10:15-12:00 Continuation of Laboratory : Coagulase test and Ancillary test for S.aureus

CETC



1:00-2:00 PRE-LAB: Enumeration of Bacillus cereus

2:00-3:00 Laboratory Proper on Enumeration of Bacillus cereus with Exercise on Sampling

CETC


3:15-4:30 Continuation of B. cereus CETC

DAY 4

8:30-9:00 Biochemical Tests for Bacillus cereus CETC

9:00-10:30 POST-LAB: Bacillus cereus CETC


10:45-12:00 POST Lab Salmonella CSM


1:00-3:00 Ancillary Tests for S. aureus CETC


3:30-4:30 Observation of plates (Pathogens) POST-LAB: Listeria monocytogenes

CSM

DAY 5

8:30-9:30 Observation of all plates and tubes CSM


9:45-10:15 Quality Assurance in the Laboratory Observation of results of workshops

CSM

10:15-12:00 Interpretation of Results CETC/CSM


1:00-3:00 Post Evaluation of Participants with Discussion Health break Post Evaluation

CETC/CSM

TOTAL no. of hours

30 hours

pathogens_training in microbiological analysis of food as of feb. 6

Documents

food microbiology

food testing laboratory

food samples

training objectives

training method

schedule of training

course objectives

training courses