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Material Safety Data Sheet

Required Products

Organisms

Organisms this product works with:

Dehydrated Culture Media

AFPA Base

Code: CM0731

A selective identification medium for the detection of Aspergillus flavus and A. parasiticus.

Typical Formula*

gm/litre

Peptone

10.0

Yeast Extract

20.0

Ferric ammonium Citrate

0.5

Dichloran

0.002

Agar

15.0

pH 6.3 ± 0.2 @ 25°C

 

* Adjusted as required to meet performance standards

CHLORAMPHENICOL SUPPLEMENT

Code: SR0078

Vial contents

SR0078E
(1 vial per 500ml medium)

SR0078H
(1 vial per 2 litres medium)

per litre

Chloramphenicol

50mg

200mg

100mg

 

Directions
Suspend 22.75g in 500ml (45.5g/l) of distilled water and heat to dissolve completely. Reconstitute one vial SR0078E per 500ml medium or one vial SR0078H per 2 litres medium, as directed. Add the vial contents to the AFPA Base. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C. Mix well and pour into sterile Petri dishes.

Description
Ideally culture media for isolating and enumerating yeasts and moulds in foods should support recovery of all viable propagules, restrict spreading moulds, inhibit bacterial growth and aid in the identification of the fungi1. AFPA Base comes close to this ideal.

Aspergillus flavus and Aspergillus parasiticus are fungi which can potentially produce highly dangerous toxic residues known as aflatoxins. They especially affect oilseeds, edible nuts and cereals in subtropical and tropical regions throughout the world due to inadequate storage conditions.

The toxins are particularly carcinogenic in humans and eating contaminated food may result in liver cancer, amongst other diseases. Liver cancer takes time to develop but the aflatoxins also act as an immuno-suppressant so that affected individuals become susceptible to a wide range of diseases. Livestock are also at risk and poultry are particularly susceptible: over 200,000 chickens died in 1994, in Andhra Pradesh, India, after eating contaminated feeds. Cattle are not so susceptible but, if they are fed on contaminated feed, the toxin may pass into the milk.

Besides endangering human health, aflatoxin contamination seriously affects the export potential of high-value commodity crops, such as edible nuts (groundnut, pistachio, cashew and almond) and spices (turmeric and chillies), which could provide an important source of income for farmers in the semi-arid tropics. Diagnosing, or even preventing, aflatoxin contamination will enable subsistence farmers to benefit from increased trade. It will also contribute to an improvement in the general health of people, often the poor, who consume contaminated foods.

The FDA has established specific guidelines on acceptable levels of aflatoxins in human food and animal feed by establishing action levels that allow for the removal of contaminated lots from the food chain. The action level for human food is 20ppb total aflatoxins, with the exception of milk which has an action level of 0.5ppb for aflatoxin M1. The action level for most feeds is also 20ppb. However, it is very difficult to accurately estimate aflatoxin concentration in a large quantity of material because of the variability associated with testing procedures; hence, the true aflatoxin concentration in a lot cannot be determined with 100% certainty.

AFPA Base is based on the formulation of Pitt, Hocking and Glenn2. It is recommended for the rapid detection and enumeration of these two species of Aspergillus, which are potential aflatoxin producers. AFPA Base is a modification of Aspergillus Differential Medium3 and shows the following advantages over this and other mycological media.

  1. Improved colour production on the reverse of the plate due to the optimal concentration of a more soluble iron salt and the addition of yeast extract. Colonies of Aspergillus flavus and Aspergillus parasiticus develop an intense yellow/orange colouration on the reverse of the colonies, and this is a differential characteristic for these species.
  2. Improved growth rate of Aspergillus flavus due to optimal balance of peptone and yeast extract.
  3. Improved inhibition of bacteria and rapidly growing fungi due to a mixture of dichloran and chloramphenicol.

Technique

  1. Process the food sample in a Stomacher using 40g in 200ml of 0.1% peptone water (Maximum Recovery Diluent - CM0733). Alternatively add the sample to 0.1% peptone water and shake periodically for 30 minutes.
  2. Dilute the sample 1:10, 1:20 and 1:40 in 0.1% peptone water.
  3. Surface plate 0.1ml of each dilution.
  4. Incubate at 30°C and examine after 42-43 hours.
  5. Count all colonies that show the reverse, yellow/orange pigmentation.
  6. Report the results as a number of colonies of Aspergillus flavus and Aspergillus parasiticus per gram of food.

    Aspergillus oryzae can produce the same yellow/orange pigmentation. It is important in the production of Asian fermented foods, particularly soy sauce, and is only rarely isolated from other sources.
    Aspergillus niger produces colonies of similar size and texture to Aspergillus flavus at 30°C. However, on the reverse the colonies may appear pale yellow but will never be yellow/orange. After 43 hours or longer incubation, colonies of Aspergillus niger remain pale yellow but begin production of black conidial heads which enables clear differentiation to be made from Aspergillus flavus.

Please note:
Moulds and fungi growing on this medium may produce mycotoxins. Therefore, as well as the normal precautions taken to avoid disseminating infection, the plates should be carefully handled and disposed of safely. This precaution would also apply to positive food samples.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

Quality control

Positive controls:

Expected results

Aspergillus flavus ATCC® 22547White mycelium, buff spores, orange underside
Aspergillus parasiticus ATCC® 28285

White mycelium, cream spores, orange underside

Negative controls:

 
Aspergillus brasiliensis ATCC® 16404 *White mycelium, black spores, yellow underside

Escherichia coli ATCC 25922 *

No growth

* This organism is available as a Culti-Loop®

References
1. Beuchat, L. R. (1984) J. Food Protection 47: 512-519
2. Pitt, J. I., Hocking, D. & Glenn, D. R. (1983) J. Appl. Bact. 54: 109-114
3. Bothast, R. J. & Fennell, D. I. (1974) Mycologia 66: 365-369
4. King, D. A., Hocking, A. D. & Pitt, J. I. (1979) J. Appl. & Environ. Microbiol. 37: 959-964
5. Jarvis, B. (1973) J. Appl. Bact. 36: 723-727

 
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