Clean room bacteriology - ambient air and bench top microbial contamination levels, and degree of environmental control in production area

Michaelsen, G. S.

English

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PROGRESS REPORT 
NSG 643 
THE BACTERIOLOGY OF "CLEAN ROOMS" 
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G. S. Michaelsen 
Project Director 
School of Public Health 
and University Health Service 
University of Minnesota 
Minneapolis, Minnesota 55455 
October 1, 1964 - March 31, 1965 
The c i t a t i o n  of names of manufacturers i n  t h i s  repor t  i s  not  t o  be construed 
as o f f i c i a l  NASA irdorsement o r  approval of  the  'ise of commercial products 2nd 
pr13 cedures referenced herein. 
https://ntrs.nasa.gov/search.jsp?R=19650017695 2020-03-17T01:36:20+00:00Z
PROGRESS REPORT 
October 1, 1964 - March 31, 1965 
NSG 643 
During the  s ix  month per iod covered by t h i s  r epor t  (10-1-64 t o  3-31-65), 
t he  major accomplishment w a s  t h e  completion of comparative r o u t i n e  sampling 
procedures i n  the  four previously se lec ted  "clean-rooms" of varying expected 
performance. 
Charac t e r i s t i c s  of Clean Rooms 
These rooms were chosen because they represent  a wide range of d i f f e r -  
ences i n  environmental cont ro ls  and personnel p r a c t i c e s  which might in f luence  
the  levels of p a r t i c u l a t e  and microbiological  contamination i n  the  a i r  and on 
surfaces .  
cate t he  f ea tu res  of i n t e r e s t  f o r  each area. 
A genera l  desc r ip t ion  of  ind iv idua l  room c h a r a c t e r i s t i c s  w i l l  indi- 
Univac Room E (Defense Aerospace Pr in ted  C i r c u i t  Area) This room is  an 
ordinary f ac to ry  a r e a  with an open b a r  j o i s t  c e i l i n g  and about 1,000 sq. f t .  
of f l o o r  space. 
i n  Room E. 
N o  s p e c i a l  precautions f o r  contamination con t ro l  are i n s t i t u t e d  
The a i r  supply f o r  t h i s  room is furnished from a general ,  o f f i c e  type 
a i r  condi t ioner  with s tandard,  2 inch f i b e r g l a s s ,  throw-away f i l t e r s .  Rate 
of a i r  flow is about t e n  changes per hour. 
Personnel w e a r  ordinary street c lo th ing ,  and from s i x  t o  n ine  ind iv idua ls  
are a c t i v e l y  engaged i n  production work i n  t h i s  room. 
service, pr imar i ly  f l o o r  mopping, are performed rout ine ly ,  with i n t e r m i t t a n t  
dus t ing  of bench tops. 
Ordinary j a n i t o r i a l  
Univac Room D (Memory Unit Manufacturing) Cer ta in  s p e c i a l  p r a c t i c e s  have 
been i n s t i t u t e d  i n  t h i s  a r ea  t o  reduce contamination by p a r t i c u l a t e s .  Between 
25 t o  28 workers are assembling components i n  Room D which occupies approximately 
I 
- 2 -  
2,000 sq. f t .  of f l o o r  space. 
The room i s  under p o s i t i v e  pressure and a i r  is  suppl ied  by a condi t ioning 
0 system which con t ro l s  t h e  temperature a t  75' F 2 5 
between l e v e l s  of 50 t o  30 per  cent.  Before en te r ing  t h e  room, a l l  a i r  is 
passed through a Cambridge Aerosolve 1/95 mechanical f i l t e r .  An "Airson" f a l s e  
c e i l i n g  system de l ive r s  forced a i r  i n to  the  a rea  a t  a rate of approximately 15 
changes pe r  hour. 
and the  r e l a t i v e  humidity 
A l l  personnel en ter ing  t h i s  area are required t o  w e a r  head covers and 
a smock over s t r e e t  c lo th ing .  
a shoe cleaner  vacuum is ava i l ab le ,  it appears t o  be used only casua l ly  and 
infrequent ly .  
washing f a c i l i t i e s  i n  t h e  dress ing  area. 
An air lock dressing a r e a  is  provided. Although 
Shoe covers a r e  not required and t h e r e  are no spec ia l  hand 
J a n i t o r i a l  s e rv i ces  include a once d a i l y  vacuuming and wet mopping of 
f l o o r s  with detergent  designated for  c l ean  room use. 
a l s o  vacuumed d a i l y  and are washed a t  one t o  th ree  month in t e rva l s .  Occasion- 
a l l y ,  a t  the  d i s c r e t i o n  of t he  workers, they may c lean  a bench top i n  t h e i r  
immediate area i f  "it looks dir ty" .  
Bench tops and legs  are 
Honeywell Room C (Component Preparation and Cleaninn A r e a )  This 750 
sq. f t .  area has i! number of features  which suggest t h a t  i t  might be somewhat 
more e f f i c i e n t  fo r  prevent ion of p a r t i c u l a t e  contamnat ion  than Room D. Work 
i n  t h i s  a r ea  is  usua l ly  c a r r i e d  on by f i v e  t o  seven people and the  t o t a l  
personnel load never exceeds ten  persons. 
The a i r  supply f o r  Room C passes through a f i b e r g l a s s  roughing f i l t e r ,  
an e l e c t r o s t a t i c  p r e c i p i t a t o r ,  an air condi t ioner  and, u l t ima te ly ,  through 
Cambridge absolu te  f i l ters .  A i r  enters  t h e  room through c e i l i n g  louvers  and 
is exhausted through wal l  vents  located near t he  f loo r .  Temperature i s  main- 
t a i n e d  a t  72' F & 5O, r e l a t i v e  humidity is  con t ro l l ed  t o  remain below 50 p e r  
- 3 -  
cent  and the  room i s  under p o s i t i v e  pressure.  
approximately 24 changes p e r  hour, with approximately 60 per  cent  make-up a i r .  
Standards f o r  t h i s  room requ i r e  t h a t  dust  counts be no higher  than 400 
p a r t i c l e s  > 5p per  cubic foot .  
Flow rate of the  system is 
S t r i c t  cont ro l  is exercised over personnel en te r ing  the  area. Except 
f o r  workers ass igned there ,  r e g i s t r a t i o n  is  required of a l l  people. 
persons must use  the  shoe cleaner  and wash t h e i r  hands before  dressing i n  t h e  
ou te r  a i r  lock. Standard dress  requirements include smocks o r  s u i t s ,  head 
covers and shoe covers. 
A l l  
J a n i t o r i a l  s e rv i ces  are c a r r i e d  out  on a r i g i d l y  maintained schedule 
and r i g i d  inspect ions ensure compliance. 
vacuumed twice and ou t s ide  passages a r e  w e t  mopped. 
During the  day s h i f t ,  a l l  f l o o r s  are 
A second s h i f t  crew is 
respons ib le  fo r  another  d a i l y  vacuuming of wal l s ,  f i x t u r e s  and hor izonta l  
sur faces ,  except bench tops and shelves.  
a l l  f l oo r s .  
by swabbing with isopropyl a lcohol  sa tura ted  sponges. 
The same s h i f t  a l s o  again wet mops 
Assembly workers are required t o  c leanse  bench tops each morning 
Honeywell Roam A (Component Assembly Area) Honeywell Clean Room A 
appears t o  be the  most sophis t ica ted  of t he  a reas  s tudied.  
worker load a r e  almost i d e n t i c a l  t o  those descr ibed f o r  Room C. 
c h a r a c t e r i s t i c s  a r e  a l s o  similar t o  those f o r  Room C, except f o r  t he  following 
important add i t iona l  f ea tu re s  : 
Floor space and 
Operating 
1. An a i r  shower is required f o r  a l l  personnel before  admittance t o  
t h e  dress ing  room a i r  lock. 
2. The a i r  handling system i s  b a s i c a l l y  t h a t  of Room C, but  i s  modified 
i n  s e v e r a l  aspec ts .  Among these is  t h e  add i t ion  of an  Aerosolve p r e - f i l t e r  
s t a g e  t o  the  a i r  stream cleansing u n i t .  Furthermore, t he  flow rate fo r  t h i s  
room i s  increased t o  30 changes pe r  hour and the  supply rece ives  33 p e r  cen t  
make-up a i r .  
1- 
- 4 -  
3 .  P a r t i c u l a t e  contamination s tandards are set  a t  200 p a r t i c l e s  > 5p per  
CU. f t .  and 25,000 p a r t i c l e s  > 0.5~ p e r  CU. f t .  
4 .  A l l  ma te r i a l s  en ter ing  t h i s  room have been cleaned previously i n  
another  area of comparable atmosphere. 
f e r r e d  i n t o  R o m  A through a i r  lock f a c i l i t i e s .  
such as penci l s ,  paper, e t c .  a r e  excluded from the  room. 
Methods 
Parts and o ther  materials are t r ans -  
P a r t i c u l a t e  shedding materials 
The rou t ine  sampling procedures, which were c a r r i e d  out  on 8 occasions 
i n  each of the  four  rooms, can be summarized a s  follows: 
1. Ambient a i r  samplinq. On each sampling v i s i t  a Reyniers s l i t  
sampler w a s  set  up a t  bench top l eve l  near t he  cen te r  of t he  room's activities. 
A series of 5 consecutive one hour samples a t  one CU. f t .  p e r  minute was 
co l l ec t ed ,  making a t o t a l  sampling volume of 300 CU. ft. p e r  day. 
were co l l ec t ed  on TSA agar and incubated 435 hours a t  32' C before counting. 
Samples 
2. Bench top su r face  s a m p l i s .  A l l  su r f ace  sampling was done with 
Rodac agar  contac t  plates. 
t h e  number of randomly chosen bench top sampling sites necessary t o  measure 
accu ra t e ly  t h e  bench top contamination i n  each room. In  Rooms A, D, and 
E, 90 s i tes  w e r e  sampled during the a.m. and 90 d i f f e r e n t  si tes during the  
p.m. f o r  a t o t a l  of 180 Rodac samples p e r  sampling day. 
v a r i a b i l i t y  i n  Room C required 150 sampling s i t e s  p e r  series o r  300 samples 
per  sampling day. 
hours a t  32' C before counting. 
Preliminary sampling da ta  were used t o  c a l c u l a t e  
An unexplained g r e a t e r  
Again, TSA agar  was used and p l a t e s  were incubated 43% 
3 .  S t a i n l e s s  steel s t r i p  contamination. S t a i n l e s s  s t e e l  s t r i p s  were 
exposed f o r  evaluat ion of f a l l - o u t  contamination i n  Rooms 'A' and 'D'. 
series cons is ted  of 42 strips which were placed i n  seve ra l  bench top loca t ions  
w i t h i n  each room. 
Each 
Six s t r i p s  from each s e r i e s  were evaluated a t  3 week 
- 5 -  
intervals f o r  a t o t a l  of 21 weeks. 
s t r i p  i n  a b o t t l e  with 25 cc  of phosphate bu f fe r  p lus  0.02% Tween 20. 
b o t t l e s  were shaken f o r  5 minutes i n  a gyrorotatory mechanical shaker;  
followed by p l a t i n g  of 5 m l  a l i q u o t s  i n  each of the  following ways: 
Five m l  p l a t ed  i n  TSA agar  and incubated ae rob ica l ly  
f o r  7 2  hours a t  32O C. 
Five m l  p l a t ed  i n  TSA agar  and incubated anaerobica l ly  
( i n  Nitrogen gas)  for  72 hours a t  32' C. 
Five m l  hea t  shocked (80' C f o r  20 minutes), then 
p l a t ed  i n  TSA agar  and incubated ae rob ica l ly  f o r  72 
hours a t  32' C. 
Five ml heat shocked (80' C for 20 minutes), then 
p l a t e d  i n  TSA agar  and incubated anaerobica l ly  for 
72 hours a t  32' C. 
Qu a l i t a t i v e  determinations. 
The ana lys i s  cons is ted  of p lac ing  each 
The 
a. 
b. 
c. 
d. 
4. From the  rou t ine  a i r  and bench top 
samples, a varying number of p l a t e s  were randomly se l ec t ed  each week fo r  
cha rac t e r i za t ion  of colonies.  The cha rac t e r i za t ions  included a morphological 
s tudy of a l l  colonies  on the  p l a t e  by smears made on 15 p lace  mi lk  s l i d e s .  
smears were G r a m  s ta ined ,  examined microscopically,  and r e s u l t s  recorded. 
The 
From t h e  s t a i n l e s s  steel s t r i p s ,  only those colonies  developing from 
hea t  shocked a l i q u o t s  were selected.  
i d e n t i f i c a t i o n  procedures. 
Resul t s  
These are being subjec ted  t o  d e t a i l e d  
The q u a n t i t a t i v e  data  from a i r  and bench top sampling i n  all four  rooms 
are summarized i n  Table I. The microbial  counts of a i r  from these  rooms r e f l e c t  
t h e  con t ro l  d i f fe rences  i n  a very p red ic t ab le  manner. For example, t h e  room 
wi th  g r e a t e s t  con t ro l  (Honeywell A )  yie lded  a mean of 0.22 col.  pe r  cu. f t . ,  
. .  
- 6 -  
while i n  the  room with least cont ro l  (Univac E) mean colony counts reached 
4.20 col .  pe r  CU. f t .  The mean a i r  counts of less than one col.  p e r  CU. f t .  
repor ted  fo r  Rooms A and C represent  extremely low b a c t e r i a l  counts f o r  
occupied areas. 
The da ta  fo r  bench tops i s  not qu i t e  as predic tab le .  The extremely odd 
d i s t r i b u t i o n  of contamination i n  Room C i s  apparent ly  respons ib le  f o r  t h e  
discrepancy as t h i s  room had a mean level of 21.0 col. pe r  Rodac p l a t e  but  a 
median l e v e l  of only 4.0 colonies  per p l a t e .  
t he  bench top l eve l s  demonstrate a r e l a t ionsh ip  t o  degree of contamination 
con t ro l  which i s  similar t o  t h a t  observed f o r  a i r  counts. These da ta  are 
now undergoing a d d i t i o n a l  s t a t i s t i c a l  ana lyses  and it may be  t h a t  t he  median 
levels w i l l  p r w e  t o  be  the s i g n i f i c a n t  es t imates  of contamination. 
I f  only the  medians are considered, 
Table I1 summarizes the  r e l a t i v e  microbial  levels de tec ted  on s t a i n l e s s  
s teel  s t r i p s  during t h e  2 1  week series i n  Rooms A and D. These da ta  provide 
evidence t h a t  t he re  is =marked contamination build-up on s t a i n l e s s  steel  
s t r i p s  over a prolonged per iod  of t i m e ,  even f o r  hea t  r e s i s t a n t  organisms. 
Indeed, i n  Clean Room A, when t h e  room was t o t a l l y  unoccupied f o r  a 
per iod  of 10 days preceding the  last s t r i p  ana lys i s ,  t he  contamination l e v e l s  
dropped v i r t u a l l y  t o  zero. 
from experiments conducted by the  Army Biological  Laboratory a t  For t  Detrick. 
Anotherpoint of i n t e r e s t  i s  the  apparently n e g l i g i b l e  d i f f e rence  i n  contamina- 
t i o n  l e v e l s  on s t r i p s  between the  clean room with r i g i d  con t ro l s  (Room A )  
and a c lean  room with r e l a t i v e l y  l i t t l e  con t ro l  (Room D). 
This information seems t o  confirm previous data 
Tables 111 and I V  i l l u s t r a t e  t h e  qualitative breakdown of microbia l  
contaminants from a i r  and bench top samples i n  a l l  four  rooms. It appears 
f r a n  these  da ta  t h a t  some differences do occur among rooms i n  the  same 
bu i ld ing  and a l s o  among buildings.  For ins tance ,  t h e  high percentage of 
- 7 -  
yeas t s  on bench tops i n  Room C were not apparent i n  t h e  a i r  of Room C or i n  
Room A. Also, t h e  very high percentages of diphtheroids  i n  Roms D and E 
d id  not  appear i n  Room A and C. HDwever, it would appear t h a t  t he  g r e a t  
major i ty  of contaminants i n  a l l  rooms were of human o r i g i n  ( G r a m  + cocc i  and 
diphtheroids)  as opposed t o  typ ica l ly  s o i l  and dus t  microbes. 
appears t h a t  the  percentage of "spore forming" b a c t e r i a  ( those which gave v isua l  
evidence of spores on G r a m  s t a i n s )  remained r e l a t i v e l y  constant  among a l l  four  
rooms (0.5 - 4.1%). 
Discuss ion  
It a l s o  
The da ta  reported here in  i l l u s t r a t e  t h a t  ambient a i r  and bench top 
microbial  contamination levels do, i n  general ,  r e f l e c t  t h e  degree of con t ro l  
a s soc ia t ed  with spacec ra f t  component production areas, a t  least i n  the  
Minnesota area during the  winter  months. However, t h e  contamination d i f f e rence  
a s soc ia t ed  with s t a i n l e s s  steel s t r i p s  exposed up t o  21 weeks i n  r e l a t i v e l y  
wel l -control led VS. r e l a t i v e l y  poorly-control led rooms appears t o  be very 
small and of l i t t l e  p r a c t i c a l  s ignif icance.  
contamination l e v e l s  do not  bu i ld  up over a prolonged exposure t i m e ,  bu t  
r e f l e c t  deposi t ion of  contamination only during a s h o r t  t i m e  preceding t h e  
ana lys i s .  This i s  wel l  i l l u s t r a t e d  by the  a n a l y s i s  of s t r i p s  from Room A 
examined a t  the  21 week i n t e r v a l  a f t e r  t h e  room had been t o t a l l y  unoccupied 
f o r  10 days, showing counts on a l l  except t h e  anaerobic  pre-heat p l a t e s  
dropping t o  zero. 
It also appears t h a t  microbia l  
It would a l s o  appear,  t h a t  even a f t e r  prolonged exposure the  percentages 
of hea t  r e s i s t a n t  microbes remain f a i r l y  constant  a t  10-15% of t o t a l  contami- 
nants  on s t r i p s ,  while i n  ambient air  and on bench tops,  spore formers make up 
less than 5% of a l l  contaminants. 
. i  
- 8 -  
Future Work 
During the  remaining months of t h e  p ro jec t  s eve ra l  areas remain t o  be 
invest igated.  
which were i s o l a t e d  from t h e  s t r i p s  during t h e  21 week series. 
are i d e n t i f i e d  they w i l l  be subjected t o  thermal death time experiments i n  a n  
at tempt  t o  p i n  down t h i s  r e l a t ionsh ip  f o r  actual c lean  room component contami- 
nants. 
s a r y  t o  a c t u a l l y  s te r i l i ze  strips of var ious types subjected t o  na tu ra l  
contamination from f a l l  ou t  and from human handling. 
An e f f o r t  w i l l  be made t o  iden t i fy  t h e  hea t  r e s i s t a n t  microbes 
Vhen these  
An e f f o r t  w i l l  a l s o  be made t o  determine the  t i m e  and temperature neces- 
Work w i l l  a l s o  be i n i t i a t e d  t o  determine t h e  f e a s i b i l i t y  of assembling 
var ious  simulated components using a sep t i c  techniques. 
I n  addi t ion ,  v e r t i c a l  laminar flow "clean rooms"wil1 be s tudied  t o  
determine microbial  contamination l eve l s  assoc ia ted  with t h i s  type of 
f aci 1 i t y  . 
-. 
. 
F 
... 
HONEYWELL (ROOM ' A ' )  
Aerobic Anaerob i e  
Week ' Before After Before After 
3 43.3 0 - - 
6 19.2 0.8 2.5 0.8 
9 16.7 3.3 12.5 0 
12 38.3 3.3 0 0 
15 4.2 1.7 4.2 3.3 
18 16.7 I 4.2 10.0 0.7 
21 w w 3.3* O* 
Mean 19.8 1.9 4.6 0.7 
Heat Heat Heat Heat 
TABLE I1 
STAINLESS STEEL STRIP SUMMARIES (COLONIES/1" x 2" STRIP) 
UNIVAC (ROOM 'D' 1 
Aerobic Anaerobic 
Before After Before After 
Heat H e a t  Heat Heat 
46.7 0 0 0 
6.7 1.7 0 0 
9.2 3.3 2.5 0 
12.5 1.7 0.8 0 
15.0 3.3 3.3 2.5 
2383 285 29.2 1.7 
36.7 9.0 16.7 2.5 
21.4 3.1 7.5 1.0 
*Room unoccupied for 10 days prior to s t r i p  analysis. 
, 
TABLE 111 
HONEYWELL SUMMARY-AIR & SURFACE 
RELATIVE OCX-ENCE OF-M€CROBIAL mPES 
Gram + cocci 
Gram - cocci or coccobacilli 
Gram +bacilli (no spores) 
Gram + bacilli (spores) 
Gram - bacilli 
Diphtheroids 
Actinomycetes 
Yeasts 
Molds 
Number of colonies examined 
ROOM A 
Air 
77.3% 
0.5 
12.2 
2.8 
6.1 
0.8 
0.3 
Bench TOP 
74.4% 
0.4 
20.6 
1.0 
1.5 
1.5 
0.1 
0.5 
362 1,428 
ROOM C 
A& Bench Top 
74.8% 69.6% 
0.2 0.1 
14.9 11.8 
4.1 0.5 
0.6 2.0 
5.0 3.6 
0.1 
__ 
I 
0.2 $2.1 
0.2 0.1 
463 2,125 
*Based on 8 sampling days (12-2-64 to 1-26-65) 
TABLE ZV 
UNIVAC SUMMARY--AIR & BENCH T O P  
REIATIVE OCCURRENCE OF MICROBIAL TYPES 
Gram + cocci 
Gram-cocci -+ coccobacilli 
Gram + bacilli (no spores) 
Gram + bacilli (with spores) 
Gram - bacilli 
Diphtheroids 
Actinomycetes 
Yeasts 
Molds 
Number of colonies, examined 
ROOM D 
A* Bench TOD 
68.5 53.7 
0.1 0.3 
2.5 4.9 
3.2 2.6 
0.1 0.2  
25.6 37.6 
0 .  0.1 
- 0 . 4  
- 0.2 
1 
786 ' 2,596 
ROOM E 
A& Bench Tor, 
49.6 3 8 . 1  
0.3 0.1 
6.1 5.4 
2.3 3 .2  
1.3 0.4 
37.1 47.0 
0.6 oms 
0.2 0.8 
2.5 4.5 
1,927 4,535 
, 
*Based on 8 sampling days (2-2-65 to 3-23-65) 


The bacteriology of ''clean rooms'' progress report, 1 oct. 1964 - 31 mar. 1965

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