CONTROL OF MICROBIOLOGICAL CONTAMINATION S u b m i t t e d by

G E R A R D J. M C G A R R I T Y Institute for Medical Research Camden, New Jersey 0 8 1 0 3 ~,,

I.

INTRODUCTION F r o m the earliest days to the present, t h e greatest needs in cell, tissue and organ culture have remained: (a) improved and standardized media; and (b) f r e e d o m f r o m microbiological contamination. No control measures will guarantee f r e e d o m f r o m c o n t a m i n a t i o n . However, certain precautions can minimize the possibility o f c o n t a m i n a t i o n . The measures listed below have p r o v e d effective w h e n followed strictly.

II.

MATERIALS Propipettes, I n s t r u m e n t a t i o n 1 Pi-pumps, Nos. F-37896 to F-37899, Bel-Art 2 Chlorophen, Roch. Germicide 3 Laminar air flow cabinets or rooms, Baker 4 and Bioquest S RTV-102 sealant, Gen. El. 6 V e l o m e t e r or t h e r m o a n e m o m e t e r , Alnor 7 T e m p e r a t u r e chart recorder, Series 1100, Fischer-Porter s Autoclave tape, No. 1222, 3M 9 Kilit spore strips, No. 12020 s Patapar paper, No. 27-144, Bristol Parchm e n t 10 Wet vacuum cleaner, with filter exhaust, Dart model, Danzig 11 Ultipore disposable filter, No. M B Y 2 0 0 1 V R A Pall 12 Uniforms or l a b o r a t o r y jackets, Angelica 13 Disposable operating r o o m caps, Busse Hosp. Disp. ~4 Pipette-Aid, D r u m m o n d Scientific is

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Make certain t h a t e d u c a t i o n o f personnel and periodic review o f aseptic procedures are carried out. Scrutinize new p r o c e d u r e s to d e t e r m i n e the risk o f c o n t a m i n a t i o n to t h e culture and o f infection to personnel.

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Examine new e q u i p m e n t to d e t e r m i n e if it might be a p r o x i m a t e source o f c o n t a m i n a t i o n . Especially i m p o r t a n t are items that contain water or moist parts and small c o m p o n e n t s t h a t are difficult to clean. Bacteria can r e p r o d u c e where p r o t e i n and moisture accumulate.

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Make sure facilities for cell culture work are o f p r o p e r design.

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Prohibit eating, drinking and smoking in the laboratory. Specific measures

S.

1. III.

PROCEDURE

A.

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Use propipettes, pi-pumps, pipette-aid or similar hand pipetting devices in order to prohibit m o u t h pipetting.

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Use quarantine/isolation facilities and techniques for handling cultures whose c o n t a m i n a t i o n status is u n k n o w n . If separate l a b o r a t o r y facilities are n o t available, questionable cultures should be handled after clean cultures.

.

Carefully disinfect w o r k surfaces between handling o f different cell cultures. There are m a n y effective disinfectants available; c h l o r o p h e n is used in this laborat o r y in a c o n c e n t r a t i o n of 1 oz per gallon o f water. C o n c e n t r a t i o n , length o f exposure and presence o f e x t r a n e o u s protein will affect the efficiency o f disinfectants. Disinfectant solutions should be discarded

General precautions .

All personnel w h o handle cell cultures, media and sterile reagents should use careful aseptic technique.

1 I n s t r u m e n t a t i o n Associates, New York, NY 2 Bel Art Products, P e q u a n n o c k , N Y 3 R o c h e s t e r G e r m i c i d e Co., R o c h e s t e r , N Y 4 Baker Co., Inc., S a n f o r d , ME s Bioquest, Inc., Cockeysville, MD 6 General Electric, Waterford, NY 7 Alnor, Inc., Chicago, IL 8 Fischer-Porter, Warminster, P A 9 3M Co., St. Paul, MN 10 Bristol P a r c h m e n t , Bristol, PA 11 Danzig F l o o r M a c h i n e Corp., D u m o n t , NJ 12pall Corp., Glen Cove, NY 13 Angelica U n i f o r m s , Inc., St. Louis, MO 14 Busse Hospital Disposables, G r e a t Neck, NY is D r u m m o n d Scientific Co., Broomall, P A

181

Use antibiotic-free media.

i m m e d i a t e l y after use. Used disinfectant m a y be a rich m e d i u m for bacterial g r o w t h . .

D.

Discard used glass and plastic ware into a pan containing d o u b l e strength disi n f e c t a n t solution. Pipettes, t u b e s and o t h e r ware m u s t be s u b m e r g e d to insure efficient disinfection. Cell culture m e d i a and o t h e r fluids m u s t be d e c a n t e d into disinfectant solution. It m a y be useful t o use separate pans for glass and plastic ware if these are handled d i f f e r e n t l y in the sterilization k i t c h e n . Pans should be t a k e n for t e r m i n a l sterilization as soon as t h e y are full or w h e n w o r k is c o m p l e t e d .

6.

Identify contaminating help d e t e r m i n e t h e source.

Testing o f l a m i n a r flow b i o h a z a r d safety cabinets .

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a.

b. .

C.

P r o m p t l y autoclave c o n t a m i n a t e d cultures as o p p o s e d t o c o n t i n u e d passage in an a t t e m p t to irradicate t h e c o n t a m i n ation. Such an a t t e m p t usually fails and leads t o infection o f o t h e r clean cell cultures.

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Store u n c o n t a m i n a t e d cell cultures in liquid nitrogen in early passage. This permits r e p e t i t i o n o f e x p e r i m e n t s w i t h a standardized cell culture and serves as a b a c k - u p in the event cultures are lost, m u t a t e or b e c o m e c o n t a m i n a t e d .

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L a m i n a r air or mass air flow cabinets or rooms .

2.

D e t e c t filter leaks b y t h e use o f an electronic particle c o u n t e r . Carefully scan t h e filter face to pinp o i n t leaks.

4.

E x a m i n e filter seal which is a f r e q u e n t site o f leaks.

5.

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Use high efficiency particulate air (HEPA) filters in laminar air f l o w or mass air flow transfer cabinets or r o o m s t o p r e v e n t airborne c o n t a m i n a t i o n during cell culture procedures, m e d i a p r e p a r a t i o n and sterility tests. These units should be c h e c k e d at least y e a r l y to insure p r o p e r filtration, air flow and air balance. Before purchase o f a cabinet or r o o m ask f o r a c o p y o f t h e u n i t ' s o p e r a t i o n a l manual. S o m e are m o s t explicit a n d helpful.

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Make sure t h a t t h e average vertical velocity inside laminar flow b i o h a z a r d safety cabinets is 100 (-+20) ft per rain

E.

Handle t h e m e d i a o f H E P A filters with care since it is e x t r e m e l y fragile and can be d a m a g e d b y even light handling. 182

If necessary, adjust t h e fan speed.

If t h e m a x i m u m velocity is still less t h a n 80 f p m , replace the H E P A filter if it is overloaded. Cabinets and mass air flow r o o m s should have direct m e a n s o f d e t e r m i n i n g t h e resistance across the HEPA, a direct m e a n s o f d e t e r m i n i n g if a n e w filter is required. H E P A filters should be changed according to m a n u f a c t u r e r ' s instructions. If infectious or p o t e n t i a l l y infectious material is used in t h e cabinet, use paraf o r m a l d e h y d e gas sterilization o f t h e old filter b e f o r e it is r e m o v e d (1). Use air v e l o c i t y to d e t e c t filter leaks and loose seals. Pass t h e nozzle o f t h e velom e t e r or t h e r m o a n e m o m e t e r over the filter face and seal. A leak in t h e filter or seal will show as a large increase in air velocity.

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Check air balance b y releasing s m o k e one inch outside the h o o d and one inch inside, and d e t e r m i n e if t h e s m o k e penetrates the air curtain. Air p a t t e r n s in t h e h o o d and air balance at t h e f r o n t o p e n i n g can be visualized b y s m o k e . Check t h e balance with the h o o d e m p t y and also with various e q u i p m e n t wares inside as it is r o u t i n e l y used.

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Do n o t use h o r i z o n t a l flow l a m i n a r flow h o o d s t h a t direct t h e air at the technician for cell culture or m i c r o biology w o r k .

Quality c o n t r o l testing and m o n i t o r i n g o f o t h e r e q u i p m e n t and supplies .

Repair small leaks with General Electric R T V - 1 0 2 sealant.

Take velocity measurements with a v e l o m e t e r or a t h e r m o a n e m o m e t e r . Most schools of engineering will have an electronic particle c o u n t e r or a t h e r m o a n e m o m e t e r to d e t e c t leaks in the filter and filter seal.

Use effective quality c o n t r o l to d e t e c t c o n t a m i n a t i o n in serum, w a t e r and m e d i a b e f o r e t h e y are grow cell cultures. F o r q u a l i t y m e t h o d s see Procedures 1 2 3 0 4 , and 75317.

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the vacuum source to remove infectious aerosols that may be generated in the trap. The trap should not become more than three-quarters full; overloading can destroy the filter.

Monitor the effectiveness of ovens and autoclaves by physical, chemical and microbiological means. a.

Make physical checks through the use of a temperature chart recorder.

b.

Use autoclave tape that undergoes color change at the temperature of sterilization.

c.

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Package 7-10 pipettes, depending on size. Wrap in Patapar paper for ready storage and quick use.

b.

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Strictly enforce effective housekeeping procedures. a.

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(3) Remove disinfectant after 5 min by means of a wet vacuum cleaner equipped with a filter exhaust. b.

Thoroughly clean water reservoirs, especially faucets, sinks and wet suction apparatus. (1) Empty water bath and turn off when not in use.

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e.

Wear clean protective clothing in the form of either full uniforms or laboratory jackets and head covering to minimize contamination by microorganisms shed by the technician or present on street clothing. This clothing should be v~orn only when working with cultures, changed regularly and not be worn outside the laboratory area.

f.

Do not allow any unnecessary activity in the immediate area during sterile procedures. This includes general traffic and talking.

Carry out periodic checking of cell cultures for microbiological contamination, including mycoplasma (see Procedure 75317).

Cell culture contamination may come from other contaminated cultures and the general environment. Contamination may originate from any source in the environment, but more frequently from the technician, unsterile supplies and media. The procedures outlined here are designed to prevent contamination from these sources. Antibiotic-containing media encourage the selection of resistant contaminants that can remain undetected unless special media and culturing techniques are used. Previous experience in this laboratory has shown that antibiotics generally were present when fastidious organisms were isolated from cell cultures. Analysis in this laboratory has shown that the major contaminants of cell cultures in antibiotic free media are microorganisms that are ubiquitous in the environment: Staphylococci, Bacillus sp., molds, pseudomonas, etc. These organisms will multiply rapidly in cell cultures and yield quick evidence of contamination, turbidity and cell destruction. In antibiotic containing media only resistant microorganisms, which are often slow growing or difficult to detect, will survive. Isolations from antibiotic containing media were characterized by more fastidious microorganisms: Hemophilus sp., mycobacteria,

(2) Autoclave sterile mop heads after each use because used moist mop heads can serve as a growth medium and incubator for microorganisms.


Store sterile supplies in dust-free cabinets.

III. DISCUSSION

Disinfect floors on a regular basis, preferably daily. (1) Obtain maximum disinfection by flooding the floor with disinfectant.

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Package sterile supplies in volumes that will be used at one sitting.

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Alternatively, insert specially prepared Kilit spore strips inside packages that are placed in the sterilizer. Upon removal, insert spore strips into broth tubes and incubate for signs of bacterial growth. Appropriate controls described by the supplier should be included.

Avoid clutter in the laboratory.

(2) Use concentrated disinfectant in liquid traps for aspirated media. This disinfectant will be diluted by aspirated media and inactivated by protein as medium is introduced. An Ultipor disposable filter or equivalent should be placed in the vacuum line between the disinfectant trap and 183

anaerobic diphtheroids, slow growing yeasts and human species of mycoplasma. Most chemical disinfectants commercially available are effective. A common difficulty encountered with disinfectants is that they are often improperly used. Concentration, as directed by the supplier, length of exposure, and presence of extraneous proteins will influence effectiveness. Proteins and other chemicals will inactivate disinfectants and may convert them to an excellent culture medium for many microorganisms. Specific implementation of some of these recommendations will vary in different laboratories, depending on design, availability of support services and nature of the work. However, these recommendations have been used successfully in many laboratories to prevent, detect and control contamination.

Safety, National Cancer Institute, Bethesda, Md. (slide cassette audio visual). 2. Fogh, J., (Ed.). 1973. Contamination in Tissue Culture. Academic Press, New York. 3. Fogh, J., N. B. Holmgren, and P. O. Ludovici. 1971. A review of cell culture contamination. In Vitro 7:26-41. 4. Coriell, L. L. 1973. Quality control measures. In: P. F. Kruse and M. K. Patterson (Eds.), Methods and Applications of Tissue Culture. Academic Press, New York. 5. McGarrity, G. J., and L. L. Coriell. 1971. Procedures to reduce contamination of cell cultures. In Vitro 6:257-265.

IV. REFERENCES

6. Barile, M. F., H. E. Hopps, M. W. Grabowski, D. B. Riggs, and R. A. Del Guidice. 1973. The identification and sources of mycoplasmas isolated from contaminated cell cultures. Ann. N. Y. Acad. Sci. 225:251-264.

1. National Cancer Institute. 1973. Formaldehyde decontamination of laminar flow biological safety cabinets. Office of Research

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