FORD FLTM EU-AT 050-2-2000 DETERMINATION OF TOTAL AEROBIC BACTERIA FUNGI AND YEAST COUNTS IN SOLUBLE OIL EMULSIONS.pdf

1、 FORD LABORATORY TEST METHOD EU-AT 050-02 Date Action Revisions 2000 08 30 Revised Editorial no technical change A. C ockman 2000 05 18 Editorial no technical change A. Cockman 1993 01 04 Printed copies are uncontrolled Page 1 of 6 Copyright 2000, Ford Global Technologies, Inc. DETERMINATION OF TOTA

2、L AEROBIC BACTERIA, FUNGI AND YEAST COUNTS, IN SOLUBLE OIL EMULSIONS Application The methods are used to determine the aerobic bacteria, fungi and yeast counts in used soluble oil emulsions. Apparatus and Materials Required Method A - Precise Method - (Total Aerobic Bacteria Count only ) Incubator C

3、apable of maintaining a temperature of 37 +/ - 1 C. Autoclave or Pressure Cooker Capable of achieving a pressure of 100 kN/m 2 at 121 C. Test Tubes 150 mm long x 12.5 mm diameter - li pless - sterile. Dropping Pipettes Graduated and sterile. Capacity 1 mL and 10 mL. Preferably glass throw away type.

4、 Petri Dishes Sterile (preferably plastic - throw away type). Nutrient Agar Prepared by dissolving 28 g of nutrient agar in 1 liter of distilled water. Source: Oxiod Ltd. Sterile Water Dettol 2 % solution Aluminum Foil FORD LABORATORY TEST METHOD EU-AT 050-02 Page 2 of 6 Copyright 2000, Ford Global

5、Technologies, Inc. Cotton Wool or Aluminum Caps For test tube seals. Formaldehyde 40 % aqueous solution. Bunsen Burner Laboratory Oven Capable of maintaining a temperature of 180 +/ - 2 C. Rotary Test Tube Mixer Steam Bath or Incubator Conditioning and Test Conditions All test values indicated herei

6、n are based on material conditioned in a controlled atmosphere of 23 +/ - 2 C and 50 +/ - 5 % relative humidity for not less than 24 h prior to testing and tested under the same conditions unless otherwise specified. Procedure A. Sterilization of Apparatus Note: All apparatus must be sterilized befo

7、re use and used only once during the determination. After use and before re - sterili zation, soak the apparatus in 2 % dettol to prevent laboratory contamination. 1. Sterilize test tubes and pipettes by wrapping in the aluminum foil and place in the oven for 1 h at 180 +/ - 2 C. Start oven off from

8、 cold. 2. After sterilization, the appa ratus should be kept in the aluminum foil until required for use. A small plug of cotton wool must be placed in the mouth piece prior to sterilization to prevent oral contamination. 3. Sterilize the nutrient agar by placing in a loosely topped bottle and place

9、 in the autoclave or pressure cooker at 100 kN/m 2 at 121 C for 15 minutes. 4. If glass Petri dishes are used, sterilize by placing in the oven at 180 C for 1 hour and then store in sealed containers. It is advisable to use sterile plastic dishes as they can be discarded after each determination. FO

10、RD LABORATORY TEST METHOD EU-AT 050-02 Page 3 of 6 Copyright 2000, Ford Global Technologies, Inc. B. Bacterial Count Note: Bacterial counts must be carried out within 6 hours of sampling. If emulsions are to be transported from the field, a sterile bottle must be used, which must be packed in a the

11、rmos flask containing ice, this will inhibit bacteria growth. 1. Pipette 9.0 mL of sterile water into each of 6 test tubes. Plug with cotton wool, or use aluminum caps. 2. Pipette 1.0 mL of the soluble oil emulsion sample into the first test tube. The end of the pipette and the top of the test tube,

12、 after removal and before replacing the plug, are passed through the bunsen flame each time to avoid contamination. 3. Mix the emulsion by using the rotary test tube mixer. 4. Using a fresh pipette, transfe r 1.0 mL of this dilution into the second test tube containing 9.0 mL of sterile water, follo

13、wing the same procedure as before, thereby achieving a 2 x tenfold dilution. 5. Continue diluting by this process until a 6 x tenfold dilution is obtained. 6. Me lt the sterile nutrient agar by placing on the steam bath. When the agar has reached a temperature of approximately 55 C, pour 10.0 mL int

14、o each of six sterile Petri dishes. The top of the nutrient agar container being passed through the bunsen flame af ter removing the stopper and before replacing the stopper. 7. Pipette 1.0 mL of each of the oil sample solution from the test tubes into the Petri dishes. Separate pipettes and flaming

15、 technique must be used. 8. Replace the top of the Petri dish and mix the emulsion into the nutrient by repeating each of the following steps five times. (a) Slide the dish backwards and forwards along the bench on a 150 mm axis without splashing. (b) Slide sideways. (c) Move the Petri dish on the b

16、ench in a clockwise ci rcular motion, and, (d) in an anti clockwise motion 9. Allow the nutrient agar to solidify and invert the plates. 10. Place in the incubator for 48 h at 37 +/ - 1 C. 11. After incubation, remove the plates and count the visible colonies in the nutrient ajar. FORD LABORATORY TE

17、ST METHOD EU-AT 050-02 Page 4 of 6 Copyright 2000, Ford Global Technologies, Inc. Evaluation Count the colonies between 40 and 400, below 40 colonies is considered inaccurate, above 400 they are considered uncountable. While counting, the plates must be examined visually to ensure that a tenfold rel

18、ationship is achieved. If this is not achieved, then contamination has taken place. The count multiplied by the dilution gives the number of bacteria per mL. Note: Destroy bacteria after counting by flooding plates with formaldehyde, and allow to react for six hours. Disposable plates should be inci

19、nerated after this treatment. Method B - Routine Methods for Total Aerobic Bacterial (Red Spot Test), Yeast and Fungi Counts Incubator Capable of maintaining a temperature of 30 +/ - 1 C. Dip Slides ( i) Biotest type GKT for aero bic bacteria count. (ii) Biotest type HS for yeast and fungi estimatio

20、n. Both types of dip slides are available as from: England Biotest - Folex Ltd. 1649 Pershore Road Birmingham B30 3DR Germany Biotest - Serum Institut GmbH 6 Frankfurt/Main 73 Post fach - 730260 France Biotest - Folex S.A.R.L. 56 Rue Jean de la Fontaine 78000 Versailles Spain Izasa S.L. Aragon 90 Ba

21、rcelona 15 Formaldehyde 40 % aqueous solution. FORD LABORATORY TEST METHOD EU-AT 050-02 Page 5 of 6 Copyright 2000, Ford Global Technologies, Inc. Procedure 1. Label the dip slide to identify system being investigated. 2. Remo ve dipslide from container. N.B. DO NOT TOUCH THE NUTRIENT MEDIUM 3. The

22、dipslide is then either: ( i) dipped into the coolant being examined or (ii) has a little of the coolant poured over it, care being taken to ensure complete coverage. Note: In the case of system being heavily contaminated with a surface layer of tramp oil, the coolant sample should be placed in a se

23、parating funnel and allowed to stand for one hour. The bottom layer should then be run into a beaker and tested as per 3 (i) or 3 ( ii). Failure to observe this precaution could result in inadequate wetting of the dipslide by the coolant, and subsequent inaccuracies. 4. Allow dipslide to drain for a

24、 few moments and return it to its container. 5. Place the container in the incubato r. Incubate for a period of: 24 hours minimum for GKT dipslides 72 hours minimum for HS dipslides 6. After incubation, remove tube from container and rate level of bacteria, fungi, or yeast when compared to standards

25、 shown in Attachment 1, and recor d results. Note: Rating is determined by the number of colonies present not their size. In the case of very heavy (confluent) growths, it should normally be reported as 10 7. If accurate results are required, the test should be repeated using a dilut ed emulsion. Di

26、lution factors should be in powers of ten, i.e. 10, 100, 1000 etc. 7. After rating destroy bacteria/fungi by filling container with formaldehyde and replacing dipslide for a period of not less than six hours. The dipslide plus the contain er should then be incinerated. Chemicals, materials, parts, and equipment referenced in this document must be used and handled properly. Each party is responsible for determining proper use and handling in its facilities. FORD LABORATORY TEST METHOD EU-AT 050-02 Page 6 of 6 Copyright 2000, Ford Global Technologies, Inc.