BASIC FOOD MICROBIOLOGY AND

ATP HYGIENE MONITORING

Presented by:
April Thessa L. Diaz

Exclusive for:
M ONDE Nissin

Date:
August 05, 2015
 COURSE OUTLINE

 Introduction to Food Microbiology

 Characteristics of Predominant Microorganisms inFood

 Sources of Microorganisms in Food

 Conventional Method and Rapid Confirmatory Testing

of Concern Pathogens

 What are our responsibilities?

 ATP as the basic and Ideal Indicator of the Invisibles

 OBJECTIVES
 To understand Food Microbiology and Its Importance
to Food Industries

 To know and identify different characteristics of

Microorganisms

 To distinguish different microorganisms and its

sources

 To understand our roles in preventing FBI and cross-

contamination

 The importance of ATP hygiene monitoring and

Indicator Rapid Testing
 INTRODUCTION TO FOOD MICROBIOLOGY

WHAT IS MICROBIOLOGY
- is the science that deals with the study of
microorganisms, including algae, bacteria, fungi,
protozoa, and viruses.

What are their functions?

- Human and animal diseases
- Soil Fertility
- Plant diseases
- Fermentation
- Food Spoilage
- Foodborne
Illnesses
 Current Status

1. Focus on study and understanding of

Pathogenic bacteria in food.

2. Development of Specific methods in Isolation

and Identification

3. Control thru sanitation to reduce

contamination brought by microorganisms

4. Interest in isolation and identification in food

industries like dairy, or food fermentation

5. Interest in immunology, medical,

pharmaceuticals and more.
E.g. Food Fermentation / Probiotics
Food Spoilage
Sensitize Vitamins
Digest Plant cellulose
Fixing Nitrogen in Plant roots
Foodborne Diseases
Application in HACCP (Food production,
processing, and preservation)
Types of Hazards:
Physical
Chemical
Biological
Biochemical
Radiological
Microbiology of unprocessed and low-
heat-processed ready-to-eat foods
Total Quality Management
(from farm to fork)
Food Safety Legislation
CHARACTERISTICS OF PREDOMINANT MICROORGANISMS
IN FOOD

Kinds of Microorganisms
1. Molds
 widely distributed in nature
 multicellular, tubular filaments, demonstrate
branching
 reproduce by fruiting bodies called spores
 capable of consuming acid
 have little heat resistance; but HRMs are
detected already
2. Yeasts
 Unicellular egg-shaped
 Smaller than molds
 Reproduce by budding
 Capable in consuming sugar and acid in
particular (Osmophilic)
3. Bacteria
 The most important and troublesome single-
celled
 Shapes: Round, Rods, Spiral, Comma
 Major Groups: Gram-negative and Gram-
positive

Cell or Bacterial Shapes

- Rods
- Round
- Spiral
- Comma
 BASIC TYPES OF BACTERIA
Illustration System
Gram-Positive Gram-Negative

Rods Spiral/
Round Borellia

S.Aureus Non-spore Spore- Facultative

Aerobe
Microaero forming forming Anaerobe
phil
Enterococcus

Pseudomonas

Lactobacillus
Listeria Salmonella
E.Coli
Shigella
Yersinia
Citrobacter
Bacillus Enterobacter
Clostridium Vibrio
Aeromonas
 Bacterial / microorganism
requirements
F.A.T.T.O.M.
Intrinsic Factors:
Food
Acidity – pH level affects enzymes and
nutrients.
Note: No known Pathogens
grow below pH 4.6
Antimicrobial
Biological structure
Oxygen – aerobic and anaerobic
Moisture - remove and/or bind moisture –
Humectants; dehydration
aw – Halophilic, Xerophilic molds,
osmophilic yeast
Extrinsic Factors:
Temperature – Psychrotrophs,
Mesophiles, Thermophiles,
Psychroduric, and Thermoduric
Relative Humidity
Gases in the environment – CO2, Ozone,
modified atmosphere packaging
Presence of other microorganisms
 SOURCES OF MICROORGANISMS
Tests and Orientation of
SOURCES Microorganisms of concern
Identification
Soil, water, establishment environment Listeria, Clostridium, Salmonella, YM, and
Escherichia
Animal feeds Listeria, YM
Animal hides E.coli O157, Salmonella, Listeria in cattle

Gastrointestinal tract Salmonella, campylobacter, E.coli

Meat, poultry, egg and egg products
O157:H7, Enterobacteriaceae
Listeria, S.aureus, B.cereus, B.anthracis,
C.Botulinum, C.perfringens, Salmonella,
Campylobacter spp., E.coli, Yersenia
enterocolitica, Brusella spp.

Seafood and marine products Vibrio spp., E.coli, Listeria Conventional culturing, Rapid
Food handlers S.aureus and all mentioned above may be testing, and Biochemical tests
present which reflects the hygiene (Gram Staining Morphology,
practices of individual including S.aureus Oxidase test, Catalase Test,
Metabolism, Motility, Coagulase
Food utensils S.aureus and all mentioned above may be
Tests)
present which reflects the GM practices of
individual including

Air and dust Listeria, YM

Dried fruits, purees, and other fruit base Yeast and mold, S.aureus, E.coli
products
Vegetables and vegetable products Depending on the ingredients of the
products. Listeria is primary concern

Globalization of food supply Major concern is the BSE, Salmonella

typhimurium, Foot and mouth disease
causing agent (coxsackievirus)

Terrorist Attack Uncertain

CONVENTIONAL METHOD VS. RAPID CONFIRMATORY TEST OF CONCERN PATHOGENS
Escherichia coli
Conventional Method Rapid Method

Conduct Presumptive Testing Conduct Presumptive Testing

Continue for Biochemical testing after positive results Continue for Biochemical testing after positive results
of presumptive test of presumptive test

Purify culture by streaking to TSA

Purify culture by streaking to TSA

Biochemical Testing using API 20

Prepare biochemical tests media for the isolate

Record and enter results after 24 hours in the API

Database for Identification
Identification at most four days and record

Send isolate and identified E.coli for DNA serotyping

to Accredited laboratory
 Staphylococcus aureus
Conventional Method and
Rapid Method

Conduct Gram-staining, Motility Test, Oxidase Test, and Catalase test

 Salmonella spp.
Conventional Method Rapid Test

Weigh 25g sample into Pre-enrichment broth and

incubate for 24H at 35 - 36°C

Option 1 (rehydrated):
Transfer 1.0mL and 0.1mL to TTBroth and RVBroth Using sterile water
and Incubate for 24H at 35-36°C Can be stored at 4°C up to 24 hours

Option 2 (autoclaved)
Can be stored at 4°C or room temp up to 4 weeks
After supplement addition, must be used within 3 hours

Steak to BSAgar, HEAgar, XLDAgar and incubate for

24H at 35-36°C

Look for Typ[ical colocnies of Salmonella and streak

into TSAgar and LIAgar and Incubate for 24H at 35-
36°C
Observe and take note of reactions in the tubes

If positive, proceed with Biochemical testing using API

If it indicates Positive Reaction for Salmonella, 20E
proceed with Biochemical testing (iMVIC Test)

Biochemical Tests will take at most 3 days

 Listeria monocytogenes
Conventional Method Rapid Test

Transfer 25g of sample to supplemented Listeria Broth Transfer 25g sample into selective enrichment broth
and incubate at 30°C for 48 H. and incubate at 30°C for 40-48H

Streak loopful in selective media and Incubate for 24-

48H at 30°C After incubation, transfer 0.4mL aliquot to test tube

Heat for 5 minutes at 100°C. Remove from heat and

Perform Identification for Positive reaction: cool to room Temperature
TSAye and Perform Catalase Test
Insert test Strip

Gram Staining

Blood Agar Inoculation

After 10 minutes, read results

Nitrate Reduction Test
1 line – Negative
2 lines - Positive
SIM Test

Drop Test

Send isolate for Serological Testing

 AGENTS OF CONCERN
Salmonella spp. – can cause severe gastroenteritis
and enteric fever

E.coli O157:H7 – is a specific strain of coliform best

indicator of possible fecal contamination derived
from human or animals.
- causes hemorrhagic
diarrhea and kidney
failure
- enterohemorrhagic
Campylobacter – Fecal contamination; isolated
from contaminated drinking water, seafoods,
vegetables, undercooked poultry, pork and beef,
and dairy products
- causes intestinal
infection and
diarrhea

Listeria monocytogenes – isolated from dairy,

meat, seafoods, and vegetables
- Causes listeriosis, fever,
muscle pain, others with
Diarrhea and other
intestinal infection
Bovine Spongiform Encephalopathy – Mad cow
disease, is a fatal neurodegenerative disease
(encephalopathy) in cattle that causes a spongy
degeneration in
the brain and
spinal cord.

Clostridium spp. – cross-contamination from soil,

feces, dust, crops, and meat
- spore-forming bacteria that can grow in the
absence of air
Staphylococcus aureus – indicator of possible
contamination of food
by a worker due to
improper handling
and poor hygiene

Norwalk and Norwalk-like Viruses - These

viruses, also known as small round structured
viruses or caliciviruses, are an important cause of
gastrointestinal illness
 FBI (FOOD BORNE ILLNESS) OUTBREAKS
• When a group of people consume the same
contaminated food and two or more of ther
individuals develop the same symptoms or illness.
• Detecting and investigating such widespread
outbreaks is a major challenge to our public
health system. This is the reason that new and
more sophisticated laboratory methods are being
developed and used by CDC and in state public
health department laboratories.

Epidemic - is used when there is an occurrence of

more cases of disease than expected in a given area
or among a specific group of people over a particular
period.

Endemic - refers to the usual prevalence of a given

disease or agent in a population or geographic area
at all times.

WHAT WE MUST DO?

Help see that the product is protected in any way
during the manufacturing process. It is our moral
and legal obligation to satisfy our customers
FIRST, LAST, and ALWAYS.

 Integrated Food Safety Approaches is GMP

 Complying with Regulations
 Preventing Catastrophes
 Improving quality and shelf life of food
 Increasing quality and confidence

Inclusions
Grounds
Plant
Equipment
Warehouse
Personnel
GOOD MANUFACTURING PRACTICES

A. Housekeeping
B. Disease Control
C. Pest Control
D. Practice the 5’s – Sort, Set in Order,
Shine, Standardize, Sustain
E. Personnel Hygiene and Practices – e.g.
Proper hand washing, Clean
Clothing, etc.
F. Water Quality and Monitoring
G. Raw Materials/Ingredients/Chemical
Evaluation
H. Evaluation of Finish products
 ATP AS THE BASIC AND IDEAL INDICATOR
OF THE INVISIBLES

No one in the food industry doubt the

value of effective cleaning or the role that ATP
(Adenosine Tri-phosphate) bioluminescence can
play in monitoring and verifying cleaning
protocols.

Importance of ATP Hygiene Monitoring and

Indicator Rapid Testing

 Limiting spread of pathogens and other

possible contaminants
 Preventing cross-contamination
 Meeting shelf life needs and claims
IMPORTANCE OF ATP HYGIENE MONITORING AND RAPID
TESTING

VI-A. What makes a Good ATP Monitoring

System?
According to Griffith et al, there are 7
conditions we need to know and evaluate in
choosing ATP Hygiene Monitoring System.

A. Choosing a System

Industry Ranking Attributes / Characteristics

1 Speed of Results
2 Accuracy of Results
3 Reproducibility of Results
4 Simplicity and Ease of Use
5 Cost per Test
6 Reliability of Equipment
Source: Industry perception of what is im portant in ATP hygiene monitoring(Griffith et al)
B. Repeatability Essential
Consistency and the very little variation
of the sum of all components in the system.
Results must not be erratic nor varies in one
sampling point.

C. Swab Reagents
A good extractant liberated the ATP from
all types of cells and simplicity of swab use with
the minimum number of steps involve in the
extraction helps to minimize variability.

D. Failure Rates
Higher failure rates from the other can
lead to a waste of time and effort as well as
leaving gaps in results making trend analysis
incomplete and more difficult with some swabs by
design being more susceptible to failure and
breakage.
E. Importance of Design
Instrumentation design, construction and
manufacture and other important factors in how
the light is captured, detected, and recorded.

Types of Instruments – advantages/disadvantages

of PMT and PD instrumentation
High stability with less or no need of regular
calibration and maintenance
Software which has the ability to easily store and
analyze the results can be a powerful help in a
root cause analysis

F. Handler / Operator
Must have a very low variation

G. Technical Service and Certificate of Validation

ATP Hygiene Monitoring Ultrasnap and Aquasnap
ATP (Adenosine-Tri-Phosphate) - is found in organic matter
from all living organisms. Monitoring ATP levels on surfaces and in
liquids is an essential indicator to overall hygiene. After cleaning or
treating, all sources of ATP should be significantly reduced.

Benefits:
Ready-to-use out of box
Easy to use and reduces user variation
Patented design produces repeatable and accurate
results
Consistent sample collection
Liquid-stable chemistry eliminates the need to reconstitute
a pellet, giving more accurate results with less variation
True and accurate results at low RLU levels
High sensitivity
Reliable trending at low ATP and low RLU levels
12-month shelf life at refrigerated temperature
6-week shelf life at 21°C
Tolerant to temperature abuse and sanitizers
Low carbon foot print
INDICATOR RAPID TEST – BACTERIAL DETECTION FOR
ENVIRONMENTAL AND FOOD TESTING

Instruments like Ensure is a one instrument –

Multiple Quality Tests that uses one instrument
platform to collect, analyze, and report data from
multiple quality indicators using the MicroSnap,
Ultrasnap, and Aquasnap.

Micro-Snap…
Total Plate Count
Coliform Count
E.coli
 The Ensure system is a simple to use, flexible
and accurate quality monitoring system for
numerous industrial applications.
Micro-Snap
- is a rapid test for identification and
enumeration of the indicated tests. It uses a novel
bioluminogenic test reaction that generates light
when enzymes that are characteristic of specific
bacteria react with specialized substrates to produce
light.
Results are available in 1 to 7 hours,
depending upon required level of sensitivity.

Benefits:
Low levels of detection
Pass / Fail result at required detection levels
are easy to understand
Quantitative results (cfu)
Qualitative Results (presence/absence)
Equivalent results to other culturing
methods
No specific sample preparation required
Independently validated
 QUESTIONS?

DAGHANG SALAMAT!