plaque assay, PFU 계산 결과

24 well plate

EPC cell
u-tube
pipet
virus (IHNV, VHS CJB)
crystal violet
PEG
methyl cellulose overlay

24 well plate 에cell seeding
clean bench 에서 virus infection (IHVN, VHS CJB)
10^1~10^8 까지 희석시킴( u-tube 8개 media 900ul, virus 100ul)
24well에  10^6~10^8 까지 희석시킨것을 넣음
PEG 처리 200ul
incubation 15min
methyl cellulose overlay
7day incubation

media 버림
crystal violet staining
30min incubation
흐르는 물에 washing
pfu 계산

결과
12 x 10^8 x 10ml = 1.2x10^10PFU/ml

discussion

이번실험은 virus를 EPC cell에 infection 시켜 pfu를 계산하는 실험이었다.
실험과정중 PEG 는 수분을 흡수하는 역할로 virus가 cell과의 interaction을 더 좋게 해준다. methyl cellulose overlay
는 다음세대의 virus에 의해 cell의 재감염을 막아 정확한 PFU 계산이 가능하게 해준다. 우리가 사용한것은 methly cellulose overlay(MC) 이지만 MC 이외에도 agar, agarose, carboxymethylcellulose, liquid medium, starch 와 같은 solid, liquid overlay들을 사용하여도 된다.
pfu 계산을 위해서는 plaque 수를 세어야 하므로 crystal violet 과 같은 vital dye 로 live cell 만을 염색시켜 plaque의 수를 센다. 이 때 cell이 제대로 seeding 되어있지 않으면 seeding되지않은 부분이 crystal violet 에 의해 염색되지 않으므로 정확한 plaque 수를 셀 수 없으므로 주의하여 seeding 한다.
우리는 virus의 희석배수를 10^3~10^8 의 virus의 plaque 수를 희석배수 별로 관찰 할 수 있었다. 이는 plaque 의 유효갯수가 10~100 개 이므로 신뢰도 높은 well 의것을 선택하여 pfu를 계산할 수 있게 함이다. 우리조의 경우 IHVN(Infectious hematopoietic necrosis virus. negative-sense single-stranded RNA virus that is a member of the Rhabdoviridae family) 를 10^8 의 희석배수로 희석한 well의 것의 PFU를 계산 하였다.








lid and liquid overlay 종류

agar, agarose , methylcellulose, carboxymethylcellulose
, liquid medium, starch

Agar overlay media.

A sterile 2% solution of agar in distilled water was prepared from purified agar
(Difco Laboratories, Detroit, Mich.) repeatedly washed with saline (0.85% NaCI). The final concentration
of agar was 1% and was prepared by mixing equal volumes of melted agar and double-strength maintenance medium at 45°C. The secondary overlay consisted of 1% agar overlay medium containing 1/10,000 (wt/vol) neutral red.

MC overlay medium.
 
The methylcellulose (MC) overlay medium was prepared as described by Schulze and Schlesinger (24). A final concentration of 0.5% MC (Methocel, 4,000 CP, Fisher Scientific Co., N.J.) in maintenance medium was used as the overlay medium.

CMC overlay medium.

The carboxymethylcellulose (CMC) overlay medium was prepared in the same manner as that for MC. The final concentration of CMC (Nutritional Biochemicals Corp., Cleveland, Ohio) in the overlay medium was also 0.5%. Liquid overlay medium. Maintenance medium, without specific antiserum, was used for the liquid overlay.

Antibody overlay medium.
 
Specific hyperimmune sera for use in antibody overlay media for Cox B-3 (bovine), Western equine encephalomyelitis (mouse), and Newcastle disease (guinea pig) viruses were obtained from the National Institute of Allergy and Infectious Diseases, Research Reference Reagents Branch, whereas hyperimmune sera to adenovirus type 3 and VSV, SIN, SFV, and Sendai viruses were prepared in rabbits by standard methods.
The antiserum was applied in maintenance medium for the antibody overlay.

JOURNAL OF CLINICAL MICROBIOLOGY, May 1977, p. 535-542

Variables Affecting Viral Plaque Formation in Microculture Plaque Assays Using Homologous Antibody in a Liquid Overlay

A. S. RANDHAWA,* G. J. STANTON, J. A. GREEN, AND S. BARON
Department of Microbiology, The University of Texas Medical Branch, Galveston, Texas 77550,* and
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of
Health, Bethesda, Maryland 20014
Received for publication 6 January 1977