Fecal Contamination in Meat • Plating Technique: Pour plate

Meat contamination Hygienic Practice and Milk Processing

Meat – refers to the fresh, chilled or frozen edible • PNS/BAFS 210:2017: Code of Hygienic Practice for
carcass including offal derived from food animals Milk
Hides or skin - post-slaughter, immediate source of • Pasteurization vs Sterilization
bacterial contamination on carcass Pasteurization – destruction of pathogenic
E. coli: Used as a marker of sanitation microorganisms
E. coli O157:H7 - enterohemorrhagic E.coli, causes Sterilization – destruction of pathogenic and spoilage
bloody diarrhea in humans microorganism
May also cause Pediatric diarrhea that can be fatal due In Milk: Reference organism for pasteurization: most
to acute kidney failure (hemolytic uremic syndrome) heat resistant pathogens in milk ➔ Coxiella burnetti and
Mycobacterium tuberculosis
Meat Inspection
Republic Act No. 9296 - THE MEAT INSPECTION CODE Organisms of concern in Milk
OF THE PHILIPPINES • Coxiella burnetti – Q fever
REPUBLIC ACT NO. 10536 – amendment to RA 9296 • E. coli O157:H7 – hemolytic uremic syndrome
National Meat Inspection Service –shall evaluate and • Listeria monocytogenes – Listeriosis (miscarriage,
classify all meat establishments following an stillbirths)
accreditation system and shall assign an accreditation • Mycobacterium tuberculosis – tuberculosis
and/or registration number to identify the • Salmonella spp. – typhoid fever, gastroenteritis
establishment and trace the products produced thereat

NMIS
Checks Meat Establishment such as Slaughterhouse,
Poultry Dressing Plant, Meat Cutting Plant, Cold Storage US and pH Standards Raw milk:
Warehouse and Meat Distribution Center in which food • US: SPC = max legal limit 100000 cfu/ml
animals or meat products are slaughtered, prepared, • pH: bulk milk = max limit 300000
processed, handled, packed or stored with National/ • Individual farm: max limit 150000
International Distribution. • Somatic cell count: US 750000; pH 400000 Pasteurized
• Issues License to operate to meat establishments US Grade A pasteurized milk = max limit 20000 cfu/ml
• Issues standards and memorandum pertaining to pH: max 50000
meat inspection and regulations
HACCP
- preventive system of applying food safety thru hazard
analysis and control measures
HACCP (Hazard Analysis Critical Control Point) is a
management system in which food safety is addressed
through the analysis and control of biological, chemical,
and physical hazards from raw material production,
procurement and handling, to manufacturing,
distribution and consumption of the finished product.
HACCP is not a stand-alone system.
When to use HACCP:
❖ International requirements
Importing countries – US, EU, Canada,
Bacterial Contamination of Milk Japan, Middle East, Australia, New
• Milk is sterile when secreted into uninfected udder Zealand, Japan and other Asian countries
• Contamination occurs during and after milking Codex, ISO and other international
(environment, equipment, handlers) Standard Agencies
• Standard Plate Count: total number of bacteria in a ❖ National regulatory requirements
milk sample that can grow and form colonies Food and Drug Administration (FDA)
• Indication of good keeping quality of milk DA Regulatory Agencies (BFAR)
• Media: Plate count agar/Standard Methods Agar
Prerequisite Programs: • Most species live as commensals on the mucosa of the
1. Facilities or Premises upper respiratory tract and lower urogenital tract.
2. Supplier Control • Pathogenicity is associated with abscess formation,
3. Production Equipment suppurative conditions, and septicemia
4. Cleaning and Sanitation (SSOP) • Virulence: enzymes, exotoxins, polysaccharide
5. Personal Hygiene capsules
6. Training of Personnel • S. suis ➔ non-pyogenic, of public health importance
7. Chemical Control causing fatal infections in pig handlers
8. Receiving, Storage and Shipping
9. Traceability and Recall Differentiation of Streptococcus spp.
10. Pest Control Differentiation can be done by the following methods:
1. Type of hemolysis – α, β, and γ
Standard Plate count: Selecting and counting plates 2. Lancefield grouping – in general, grouping for β
• Average number of colonies x Dilution Factor volume hemolytic
plated Streptococcus; presence of C-substance
Selecting and counting plates 3. Biochemical testing – used for differentiation of α and
Count promptly after incubation or store at 4 Celsius for γ hemolytic strains
not more than 24 hr. 4. Molecular Methods – PCR based methods
Record results of control plates.
• Use the following as a guide: Blood Agar
A. NORMAL (plates with 25-250 colonies or <25 Blood agar – an enriched media that uses defibrinated
colonies): mammalian blood to enrich a basal medium such as
Select spreader free plate(s). Count all colonies, tryptic soy agar
including those of pinpoint size on selected plate(s). ➔ blood is added to the base to provide additional
B. CROWDED PLATES (more than 250 colonies): Do not growth factors required for fastidious organisms.
record counts on crowded plates from the highest ➔ Blood agar is mostly used for the cultivation of
dilution as too numerous to count (TNTC). If the number pathogenic organisms that can produce extracellular
of colonies per plate exceeds 250, count colonies in enzymes causing hemolysis of the blood
those portions of the plate that are representative of
colony distribution and calculate the Types of hemolysis on Blood Agar
Estimated Standard Plate Count (ESPC) from these Alpha hemolysis - Alpha-hemolysis is partial or
counts incomplete hemolysis indicated by Greenish or hazy
C. SPREADERS. There are three distinct types of zones around colonies.
spreading colonies. (1) a chain of colonies, not too • Alpha hemolysis is caused by hydrogen peroxide
distinctly separated, that appears to be caused by produced by the bacterium, oxidizing hemoglobin to
disintegration of the bacterial clump. (2) one that green methemoglobin
develops in the film of water between the agar and the Exemplified by S. pneumoniae and other Viridans group
bottom of the dish. (3) one that forms in the film of of Streptococcus such as S. viridans
water at the edge of or on the surface of the agar • Beta-hemolysis is complete hemolysis indicated by
clear zones around colonies. Exemplified by S. pyogenes
Streptococcus ssp - CAMP TEST and S. agalactiae, and Lancefield Group
Characteristics of Streptococcus spp ➔Beta-hemolytic streptococci are generally more
• Gram-positive cocci in chains pathogenic than alpha-hemolytic
• Fastidious - requiring enriched media compared to Lancefield group:
common bacteria • Group A Streptococcus (GAS): S. pyogenes
• Small usually hemolytic, translucent colonies
➔Streptolysin, an exotoxin, is produced by the bacteria,
• Catalase-negative
which causes the complete lysis of red blood cells
• Facultative anaerobes, usually non-motile
• Group B Strep (GBS): S. agalactiae - Colonies of group
• Commensals on mucous membranes
B streptococci often have less pronounced zones of
• Susceptible to desiccation
beta-hemolysis than do other beta-hemolytic
• Cause pyogenic infections
streptococci
Habitat and Pathogenicity of Streptococcus sp.
Lancefield grouping
• System of classification developed by Rebecca
Lancefield
• a serological method of classification based on the
group-specific C-substance
• C-substance ➔ a cell-wall polysaccharide antigen, that CAMP test Principle
differs between species or groups of species and is • Named after Christie, Atkins, and Munch-Peterson
widely used to classify clinical isolates of Streptococcus. CAMP test detects the production of a diffusible,
• Divides Streptococci into 20 groups from A to V (w/o I thermostable, extracellular protein known as CAMP
and J) factor, produced by Group
➔Uses a precipitin test based on extractable group- B Streptococcus.
specific carbohydrate antigens The CAMP factor acts synergistically with the beta lysin
➔Latex agglutination test. Specific C-substance antisera produced by Staphylococcus aureus to produce a zone
for groups A to G (except group E) of enhanced lysis of sheep or bovine erythrocytes

Types of hemolysis on Blood Agar

Gamma - hemolysis denotes no observable changes
in the blood agar.
Ex. Enterococcus faecalis and E. faecium (formerly
Group D streptococci)

Serological Tests
• Optochin test – S. pneumoniae is optochin sensitive
other viridans group are resistant
• Bile solubility test - S. pneumoniae is soluble other a-
hemolytic streptococcus are resistant

Biochemical Test
• Bacitracin test - Streptococcus pyogenes is inhibited Esculin test
by bacitracin other beta-hemolytic strains are not Test for the ability to hydrolyze esculin to esculetin;
• CAMP test detection of ß-hemolytic streptococci presence of esculinase enzyme. Esculetin reacts with
Group B, GBS) Iron salt to produce black color compound
S. agalactiae Group D strep: black colonies
S. Agalactiae – white to blue colonies
Bovine Mastitis
• Costly disease in Dairy Cows
• Inflammation of mammary glands that occurs as an
immune response to bacterial invasion
➔Streptococcus agalactiae S.
dysgalactiae and S. uberis are principal pathogens. E.
faecalis is less common
• Spreads in between milking by contaminated teacup,
milker's hands, cloth towels

Diagnosis (+/-)
• Clinical signs: Swelling, pain, redness, and warm
Special Culture Technique (Candle Jar Technique)
udder, clots of milk
Classification of Bacteria based on Oxygen Requirement
• Blood agar, CAMP test, Esculin Test, Growth on MAC,
• Aerobes – grow in the presence of oxygen
Lancefield
• Anaerobes – grow in absence of oxygen
• Facultative Anaerobes – grow in presence or absence
of oxygen
• Microaerophiles – requires low oxygen concentration (c) Reproductive (fertile) hyphae: aerial hyphae carrying
than atmospheric concentration the reproductive structure (spore).
Bacteria requiring a high concentration of Carbon (d) Coenocytic hyphae: Non-septate (no cross walls)
dioxide, higher than atmospheric concentration hyphae, which allow uninterrupted flow of protoplasm
• Capnophiles ➔ usually human and animal pathogens and nuclei seen in Zygomycetes.
• Many microaerophiles are capnophiles (e) Septate hyphae: contains cross walls (ascomycetes,
Capnophiles basidiomycetes, Deuteromycetes)
• Neisseria gonorrhoeae
• N. meningitidis
• Haemophilus influenzae – upper respiratory tract
infection
• S. pneumoniae - pneumonia
• Brucella abortus – cattle abortion
• Actinobacillus spp. – pleuropneumonia in pigs
• Campylobacter jejuni – abortion, diarrhea
Actinobacillus
Techniques to increase CO2:
• Carbon dioxide incubator
• Gas generating system
• Candle Jar technique

Fungi: Morphology and Lab Diagnosis

Fungi: General Characteristics
Classification by septation
• Fungi are eukaryotic, heterogeneous, unicellular to
A. Generally Nonseptate (coenocytic) Mycelia
filamentous and heterotrophic organisms that lack
chlorophyll. I. ZYGOMYCETES ➔Rhizopus, Mucor, Rhizomucor
• Cell wall is composed mainly of chitin, some mannans B. Septate Mycelia
and other polysaccharides; plasma membrane contains II. ASCOMYCETES - Aspergillus Trichophyton, Penicillium
ergosterol III. BASIDIOMYCETES - Cryptococcus
• main storage form of Carbo: Glycogen IV. DEUTEROMYCETES
• Mode of reproduction: asexual (budding or asexual Classification
spores) and/or sexual (sexual spores) A. Perfect Fungi - can reproduce by both sexually
Fungi: Forms produced spores and asexual spores
Fungi that occur in both yeast and mold forms are I. ZYGOMYCETES - sporangiospores, zygospore
termed dimorphic fungi. II. ASCOMYCETES - conidia, ascospore
1. Yeast – unicellular fungi* whose vegetative growth III. BASIDIOMYCETES – conidia, basidiospore
predominantly results from budding or fission and B. Imperfect Fungi - can reproduce only by asexual
whose sexual states are not formed within or in a spores
fruiting body. IV. DEUTEROMYCETES
• May form pseudohyphae (chains of elongated Other asexual spores
ellipsoidal cells with constriction) and/or true hyphae • Blastospores (emerging by budding from the parent
2. Molds – Multicellular, filamentous fungi consisting of cell),
a mass of branched or branching hyphae which is either • Arthrospores (formed by fragmentation of hyphae
submerged or aerial, and vegetative or fertile. into single cells)
• Reproduce chiefly using asexual spores, although • Chlamydospores (thick-walled spores formed in
some form sexual spores in the so-called perfect stage. response to unfavorable environmental conditions)
• The whole body of mold is known as thallus
Laboratory Diagnosis of Fungal Infections
Types of hyphae Types of Specimen:
(a) Vegetative hyphae: They penetrate the artificial • Skin , hair , & nails (for dermatophytes)
medium to absorb the nutrients. • Sputum , exudates, urine , blood, C.S.F, tissue biopsies
(b) Aerial hyphae: They grow above the surface of the (usually for subcutaneous and systemic mycoses)
artificial medium.
Fungal culture media widely used method for staining and observing fungi
The following media are used for the primary recovery because it’s easy and fast.
of saprophytic & dimorphic fungi Adhesive tape method - Disadvantages: Only the
➔Sabouraud dextrose agar – main media for clinical superficial structures of the fungi tend to stick to the
use tape. Advantage: The fungal morphology appears intact
➔Brain-heart infusion agar – fastidious fungi compared to the Tease Method
B.dermatitidis, H. capsulatum Slide culture technique (Riddel technique) - Best
Isolation and identification of dermatophytes technique to use for microscopic study of molds; to
➔ Mycosel avoid disturbing the arrangement of the fungus
Culture Media structures
• Sabouraud Dextrose Agar (SDA) – media for growth of KOH mount and Fungal Stains
yeast and molds • KOH mount - primary screening method for
• Adjusted to pH 5.0 which favors growth of mycotic identifying fungal
agents, discourages growth of bacteria elements in clinical samples
• SDA with antibiotics – makes SDA selective by the KOH – as a strong alkali with the aid of gentle
addition of cycloheximide and chloramphenicol. Non- heating digest and dissolve tissue materials in hair,
pathogenic fungi are inhibited, while pathogenic are skin, or sputum samples
encouraged to grow. Bacteria are inhibited by Use to diagnose dermatophytosis, mucormycosis,
chloramphenicol; aseptate molds are inhibited by infection from Blastomyces dermatitidis
cycloheximide • Lactophenol blue - a widely used routine laboratory
• The yeast phase grows at 37 C, while the mold phase staining technique
grows at 25 C Contains the following active agents:
Differential media Phenol – acts as disinfectant and kills fungus
Corn meal agar with Tween 80 &Trypan blue for Glycerol – prevents drying of specimen
identification of C. albicans Cotton blue – stains outer wall of fungus blue,
*Potato dextrose agar for demonstration of pigment background appears light blue
produced by T. rubrum Lactic acid – preserves fungal morphology
*Niger seed agar for identification of C. neoformans. Performed usually in two ways: tease mount or
*Urea medium differentiation of T. rubrum and T. adhesive tape
mentagrophytes (+ for urease, media turns red) • India Ink - Acidic dye use in negative staining,
Temperature and Incubation requirement especially for capsules
◆ Temperature requirement Used to detect C. neoformans which causes meningitis
Majority of fungi – 37°C The capsule appears as a clear halo against the dark
Superficial mycosis – 30°C background
Dimorphic fungi – 25°C & 37°C • Periodic acid-Schiff stain or PAS - a staining technique
◆ Incubation time that identifies the presence of glycogen,
At least 4 weeks polysaccharides, and mucin in the tissue
Usually positive cultures are obtained in 7-10 days PAS stains the nuclei blue and fungi in deep pink or
Candida & Aspergillus - 24 to 72 hrs magenta color.
Colony morphology PAS stain is useful for identifying Cryptococcus
Observe for the following: neoformans, Histoplasma capsulatum, Aspergillus
➔ Texture fumigatus, and Blastomyces in tissue samples because
their cell walls and capsules contain a significant
➔Pigmentation (surface and reverse side)
amount of carbohydrates.
➔Rate of growth
➔Folds and ridges on the surface Important Genera of Yeasts in Veterinary Medicine
Identification of fungal cultures
➔ Cryptococcus - basidiomycetous, dimorphic fungus
• Colony morphology – color, texture, pigment
that often has a very thick capsule
production
Cryptococcus neoformans and Cryptococcus gattii
Fungal Microscopy
• Cryptococcosis – associated with ulcerative lesions
Direct Mount/Tease method - Disadvantage: The intact
affecting upper respiratory tract (including nasal
morphology will not be seen Advantages: It is the most
sinuses), the central nervous system (meninges), and
the eye (chorioretinitis). Non-contagious
• Infection is through the respiratory route➔ Inhalation
of spores
• Reported as the most common systemic mycosis of
cats: facial lesions are glistening and gelatinous
• Also reported in many veterinary species including
canids, equids, camelids, ruminants, ferrets, birds, and
marsupials, among others.
Malassezia pachydermatis – zoophilic, basidiomycetous Detection of Mycotoxins:
yeast that is found as normal flora of skin and ear canals • Immunochemical technique – ELISA, immuno-based
➔an outgrowth of this opportunistic fungus aggravates Flow cytometry
the disease processes already in progress • Chromatographic techniques – TLC, HPLC
➔Associated disease: otitis externa and dermatitis
Impact of Mycotoxins on Animal Health:
➔Detection via direct swab from ear canals with otitis;
• Affects digestive system, liver and immune systems
a high number of the mcg indicates the presence of the
• Reduce animal productivity
yeast
• Poultry and pigs are at higher risk due to consumption
➔more observed in dogs than in cats of feeds from cereals
➔Assimilates carbon of glucose and d-mannitol, but • Monogastric animals are more prone to mycotoxins
does not ferment carbohydrates than ruminants
Candida albicans - parasitic yeast which inhabits the • Some aflatoxin can be excreted in Milk
mucous membranes of most mammals ➔ increase exposure to children
➔are normal flora of the gastrointestinal and urogenital Aflatoxins: retarded growth, liver damage,
tracts and skin of mammals and birds. However, C. immunosuppression, and pale gills may occur in fish
albicans can invade any organ of the body and cause Ochratoxin A: causes deformation of fish, delays
disease. growth, targets kidneys in companion animals
➔Disease: Candidiasis
➔ thrush in birds, infertility and abortion in horses, Dermatophytes
ulcerative lesions in swine • Dermatophytes are molds capable of parasitizing only
Mycotoxin Producing Fungi keratinized epidermal structures: superficial skin, hair,
• Mycotoxins - secondary metabolites of some species feathers, horns, hooves, claws, and nails.
of mold fungi • Those that have a sexual reproductive phase belong to
➔They are present in many foods consumed by animals ascomycetes.
and they most often contaminate products of plant and • produce septate, branching hyphae
animal origin. • Dermatophyte infections are called ringworm or
➔difficult to remove due to their natural resistance to dermatophytoses.
mechanical, thermal, and chemical factors. • Major genera of dermatophytes: Trichophyton,
Maximum Permissible levels are regulated worldwide. Epidermophyton and Microsporum
Maize is the most common cereal infested with molds • Trichophyton ➔ affects skin, hair, nails
and is commonly used in animal feeds • Epidermophyton (only in humans) ➔ does not affect
Diseases produced by mycotoxin are called hair
mycotoxicoses • Microsporum ➔ does not affect nails Disease:
Molds often responsible for the production of ringworm clinically known as tinea
mycotoxin are the ff Ascomycetous molds: Symptoms: irritation, erythema (redness of the skin),
Fusarium spp –sickle-shaped macroconidia edema (swelling), and vesiculation (formation of the
Aspergillus spp – large vesicle vesicle or cyst)
Penicillium spp - Branched or unbranched
conidiophores separate into metulae; Flask-like phialide Wood’s lamp
cells form at the end of such metulae, which gives rise A small hand-held UV light is used to diagnose and
to conidia manage superficial cutaneous fungal infections. It is
Penicillium and Aspergillus used for the examination of the scalp and ringworm
infection. Most of the infected hair fluorescence when
exposed to ultraviolet light
Detection of Coliforms in water by MPN method Multiple fermentation Tube Technique method: Protocol
Phil Law on Water 1. The Presumptive test, which screens for potential
• Phil Clean Water Act of 2004 (RA 9275) – aims to presence of coliforms
protect the country’s water bodies from pollution from 2. The Confirmed test, which verifies the initial findings.
land-based sources 3. The Completed test, which finalizes the results.
• DENR – the primary government agency responsible
for the implementation of RA 9275 1. Presumptive test
Purpose:
Coliform Group • determine the presence of coliform bacteria
• aerobic and facultatively anaerobic • obtain the possible number (MPN)
• gram-negative • provide optimum conditions for the growth
• non-sporing rods (resuscitation) of desired organisms especially those in
• ferment lactose low numbers and injured cells
• produce acid and gas within 48 hours at 350C of • determines whether further testing will be conducted
incubation thus, saving time and effort
TYPES OF COLIFORMS
1. Fecal coliforms – thrive in gastrointestinal tract of Lauryl Sulfate Tryptose Broth (LST)
humans and warm-blooded animals • Selective enrichment culture medium
- includes the genera of Escherichia ➢ has phosphate buffer that prevents drastic change in
2. Saprophytic coliforms – non-intestinal in origin i.e. pH during lactose fermentation (acid production)
soil ➢lauryl sulfate largely inhibits the growth of non-
Significance of the presence of coliforms coliform bacteria
1. index of general sanitation ➢contain high amount of tryptose or trypticase
➢ indicator of fecal pollution and efficiency of (pancreatic digest of casein) and lactose ensuring the
wastewater treatment, processing methods high nutrient quality of this medium, thus it ensures
(pasteurization, etc.) rapid growth and increased in gas production of even
2. indicates the presence of pathogens “slow fermenting” coliforms
➢ e.g enteropathogenic E. coli (ETEC) can cause Results:
gastroenteritis in domestic animal and humans. 1) Positive lactose fermenter – gas production (CO2) in
MPN method the Durham tube
Principle 2) Negative lactose fermenter – no gas evolution
The most probable number (MPN) technique is a Obtain MPN value for lactose fermenters
method for estimating the number of bacteria in a food
or water sample. Confirmatory test
➔In this technique, replicate portions of the original • Confirms whether the positive presumptive test or
sample are cultured to determine the presence or lactose fermenters as coliforms or not
absence of microorganisms in each portion (serial Medium:
dilutions). 2% Brilliant Green Lactose Bile Broth (BGLB)
most commonly applied for quality testing of water •selective enrichment, enumeration (MPN) and for the
to ensure whether the water is safe or not in terms of confirmation of the presumptive test
bacteria present in it. •inhibit the growth of undesired microbial flora
APHA AND AWWA including lactose-degrading clostridia (e.g. Clostridium
Based on certain probability formula, which is an perfringens
estimate of the mean density of coliforms present in the Results:
sample. 1) Positive tube – gas production (CO2) inthe Durham
Coliforms – include facultative anaerobic, gram -, non- tube thus, coliform org.
spore forming, rod shaped bacteria that ferments 2) Negative tube – no gas evolution
lactose with gas and acid production within 48hrs at Obtain the MPN for the positive tubes
35C ➢ to determine the MPN value of coliform org per quantity
Total coliforms: includes thermotolerant coliforms and of the sample
bacteria of fecal origin and environmental sources
Fecal coliform: thermotolerant coliforms nearly always Completed test
indicate fecal contamination Eosin Methylene Blue Agar (EMB)
-selective and differential medium
- differentiates lactose-fermenting from lactose-non-
fermenting bacteria through acid production
- the dyes Eosin-Y and methylene blue inhibits the
growth of gram-positive organisms and permits the
differentiation of enteric coliforms
- E. coli colonies are blue-black with greenish metallic
sheen caused by their production of large quantities of
acid that eventually precipitates out the dyes on the
surface
Enterobacter aerogenes produce colonies with thick,
mucoid and grey-brown centers
Standards: >1.1MPN/100mL
10,000MPN/100mL waste water