Isolation and enumeration of yeasts (excluding probiotic yeast), moulds, Dematiaceae and aerobic/mesophilic bacteria from animal feed samples
2.0 SCOPE:
This SOP is applicable to the Animal House Facility.
3.0 RESPONSIBILITY:
In-charge Animal house shall collect the urine sample and will maintain the record.
In-charge Microbiology is responsible for overall compliance of this procedure.
4.0 INTRODUCTION:
3.0 RESPONSIBILITY:
In-charge Animal house shall collect the urine sample and will maintain the record.
In-charge Microbiology is responsible for overall compliance of this procedure.
4.0 INTRODUCTION:
Colonies of yeasts, moulds, Dematiaceae and aerobic/mesophilic bacteria detected in animal feeds may be diagnostically differentiated as indicator micro-organisms and data used regarding their growth may be used to describe the feed quality as regards its condition as microbiologically sound and unspoilt. Yeast can be described as round to oval unicellular fungi which reproduce by budding. Some are able to produce pseudohyphae (chains of elongated budding cells) but only a few are able to produce true hyphae. Yeasts are characterized, classified and identified by their morphology and biochemical laboratory tests.
Yeasts are widely distributed in the environment and are part of the normal flora of man and animals. Yeasts may contaminate a wide variety of animal feeds (including dry feeds) and may cause spoilage of silage. A number of microbial products may be added to animal feeds to provide beneficial effects on health and production but are not attributed with a nutritional role (probiotics). Yeast cultures may be used as probiotics with Candida pintolopesii, Candida saitoana and Saccharomyces cerevisiae commonly used. The presence of such probiotics should be established with the animal feed supplier/manufacturer if detection of yeasts is requested.
C. albicans is commonly associated with animal disease and grows as a budding yeast cell on many different agar cultures at a wide variety of temperatures and differing pH. They produce distinctive convex colonies with a shiny waxy appearance.
Mouldy animal feed does not necessarily contain dangerous mould poisons or mycotox- ins but the presence of considerable mould in animal feed will adversely affect production and health of animals feeding on it. Digestibility may be affected for ruminants and energy values will be reduced. Dematiaceae are saprophytic fungi which are widely distributed in soil, water and decaying vegetable material. Mesophilic bacteria are bacteria which have an optimum temperature of 20–45 °C and typically are food contaminants (including animal feeds).
This procedure applies to all samples submitted to the microbiology laboratory for detection of Yeasts (excluding probiotic yeasts), moulds, Dematiaceae and aerobic/meso- philic bacteria in animal feeds.
5.0 PROCEDURE:
Add the required sample quantity (± 0.1 g or 0.1 mL) to the required volume of peptone water and shake for 20 min on a horizontal shaker at 120–180 rpm. If foaming is noted a few drops of suitable silicone antifoaming agent may be added to the solution.
· Alternatively the sample and diluents may be placed in a stomacher bag and after being left for 10–15 min, it may be treated in the stomacher for 3–5 min. Liquid feeds may be treated for a minimum of three min without the holding time of 10–15 min
· If appropriate the sample may be treated with a blender in a sterile beaker. First at 5 000 U/min for one minute followed by 8 000–10 000 U/min for five min.
· The procedure noted above may also be used to enumerate aerobic/mesophilic bacteria in feed samples by substituting DRBC and DG18 with tryptose agar which includes 2,3,5- triphenyltetrazolium chloride (TTC). The incubation temperature should be changed to 30 ± 1 °C for two days. TTC is reduced to red formazan by bacteria considered as indicator micro-organisms of spoilage in animal feed. After three days only colonies with a reduction of TTC should be counted.
· Bacteria which are considered indicators of spoilage in animal feed include:
ü Yellow coloured bacteria;
ü Pseudomanas spp.;
ü Enterobacteriaceae;
ü Bacillus spp.;
ü Staphylococci;
ü Micrococci;
ü Streptomycetes.
· Aerobic/mesophilic bacteria may be reported as the number of colony forming units (cfu) per gram or millilitre of feed by multiplying the number of colonies by the respective dilution factor. Bacteria are usually reported as 106 cfu/g or cfu/mL.
6.0 PRECAUTIONS:
· Protective laboratory coats, gowns, smocks, gloves or uniforms designated for lab use will be worn while in the laboratory.
· All infectious agent cultures, stocks, animal carcasses and/or tissues, sharps used to inoculate animals, etc. and bedding from infected animals will be disposed of as hazardous waste (i.e. put in an appropriately labeled red biohazard bag, and decontaminated by autoclaving.
7.0 REFERENCES:
· ISO 7218:2007. Microbiology of food and animal feeding stuffs – general requirements and guidance for microbiological examinations. Geneva, Switzerland.
· ISO 21527-2:2008. Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of yeasts and moulds. Part 2: Colony count technique in products with water activity less than or equal to 0.95. Geneva, Switzerland.
8.0 ABBREVATIONS:
SOP: Standard Operating Procedure
cfu: colony forming unit
END OF THE DOCUMENT
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1. List of In-vivo laboratory Vendors (Capex)
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3. List of All SOPs and Documents for Laboratory Instruments and Equipment
4. List of All SOPs and Documents for Animal House Facility
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