Microbiology For Utilities And Applications Of Aseptic Processing

Disinfection

Disinfection and cleaning are considered as one of the most crucial activities in healthcare and companies dealing with manufacture of pharmaceuticals and medical devices. They are vital to prevent microbial infection in hospitals and particularly during surgeries and post-operative care. In order to control or eliminate the contaminants microbiological in nature that may occur in work place., it is required to put in place a system of cleaning under control environment controlled followed by disinfection/or Sterilization processes.( David, J.R. 2012. Handbook of aseptic processing and packaging Vol. 4).

Disinfection resists microbial growth using chemical agents chemicals that are toxic in nature s such as bleaches, phenolic compounds and ethanol), physical agents (UV light and mild heat treatments (62 to 85 for a number of minutes for instance). Disinfection may also involve the destruction of microbial cells and viruses, however microbial spores are generally not damaged by the common domestic disinfectants. Chemical agents meant for destroying microbial spores, particularly bacterial endospores are termed as “HIGH LEVEL  

Critical Issues for Effective Disinfection

There are a number of considerations in order to disinfect an object.

 (1)  Disinfectant concentration

 (2) The time for the disinfection process.

 (3)  Residues or soils on the object being disinfected

 (4) The temperature required for the process of disinfection.

In general, concentration of disinfectants used are mentioned in the specification  by the manufacturers of the disinfectants and in their Standard Operating Procedures (SOPs). The minimum tie required to carry out the disinfection may also be recommended by the manufacturer. The temperature of the disinfection will be room temperature in this experimental session, however some companies may require that high temperature disinfection be used in some circumstances such as when disinfecting areas where blood products have been stored or handled, Spore-forming microorganisms, both bacterial and fungal, may withstand disinfection for extended periods of lime and in some cases concentrated disinfectants for lengthy periods of time may be required to destroy spores.

To analyze the effects of disinfectants like anti bacterial soap, savlon,70 % IPA, Hibiserub, S Aureus, E Coli using  laboratory strain of E. coli and S. aureus. 

Materials  Lab Manual is referred

Methods   Lab Manual is referred.

Results

Table 1 is referred for detailed analysis. 

Plate/Organism	Disinfectant (Antibiotic Control )	Zone of  clearance (Yes /NO )	Width of Zone
S.aureus	1.      Control	No	0 mm
2.   Anti Bacterial  Soap	Yes	20 mm
3.       70 % IPA	Yes	2mm
4.       S. Aureus	No	0mm
5.          Savlon	Yes	12mm
6.        Hibiserub	Yes	12 mm
E.Coli	1.         E.coli	No	0 mm
2.         70 % IPA	Yes	3mm
3.         Savlon	Yes	3mm
4.         Control	No	0mm
5.      Anti Bacterial Soap	Yes	7mm
6.       Hibiserub	Yes	6mm

TABLE -1 Showing effectiveness of six types of disinfectants using s aureus and E Coli as culture medium

Discussion : Table 1 shows the degree of effectiveness of the disinfectants under similar conditions using two culture media S Aureus and E Coli.. The width of zone determines the effectiveness of the disinfectants. The more the diameter more will be the effectiveness .Using plate S Aureus Anti bacterial Soap is showing width of 20 mm which is the highest. Savion, Hibiserub, is also showing good results, whereas control, S Aureus does not show any result. Similarly using E-Coli as medium , though the effectiveness is comparatively less Anti Bacterial soap and Hibiserub are highly effective. Hibiserub contains chlorhexidine gluconate which kills  bacteria, yeasts, some fungi and viruses. Antibacterial soaps contain triclosan   which inhibits growth of various bacteria however UFDA  has banned usage  of  nineteen  chemicals which are used in manufacture of  anti bacterial soap including triclosan..The active ingredients of savlon are cetrimide and chlorohexidine gluconate used for prevention of infection in small cuts and burns . (Tripathi, K.D., 2013, 900)

Critical Issues for Effective Disinfection

Conclusion :  The experiment  on effectiveness of disinfectant was conducted as per the lab manual. The results are in conformity with the expected results. The effectiveness of biological and chemical disinfectants have been tested. The chemical disinfectants are more powerful and their effectiveness has been tested positive.  

Autoclave is one of the important instrument which is simple in operation being used  in microbiology laboratories . It works like a pressure cooker. The same concept is used in autoclave to sterilize different media .In  production units autoclave is used  to sterilize different equipments. It is important that this process should be under control environment  and  the material should be completely sterilized. This is also called steam sterilizer. The experiment will show  the use of biological and chemical indicators for the validation study on an autoclave. (Tripathi, K.D., 2013. 900)

S  stearothermophilus spores in the form of either strips or ampules  placed in different  positions of  the load. B. stearothermophilus spores are one of the   microbes having high resistivity  to  steam sterilization (optimum growth temperature  50- 60°C and  survive 5 minutes at a temperature of   121°C). When the autoclave operation is done , the  strips were  hygienically  transferred to a spore strip broth., then  incubated for 55°C .  We can say that there is a growth  if turbidity is increased (spore strip broth) and there is a change of colour (ampoules). Thus, there is no growth we can conclude that  the  control strips were satisfactory  , all the spores were killed  and the sterilization was through  in that part of autoclave. Since the quantity of spores in  Browne’s  tube ,a biological indicator,  is already  known , we are in a position to  analyze  and quantify the extent of sterilization.

: Browne’s tubes a chemical indicator  is a patented product, it contains a chemical the colour of the chemical changes from brown to green once a temperature of 121°C  is attained inside the autoclave.

Autoclave tape: Autoclave adhesive tape is used as the third indicator ,usually lead carbonate based ,in this experiment. Autoclave tape contains a colour indicator  that  turns black when it comes in contact with steam. This however does not indicate whether the required temperature is reached or not. 

To determine whether the functioning of autoclave is proper to sterilize using spore strips containing chemical indicator B. stearothermophilus spores, a chemical indicator Browne’s  tube and Autoclave tape.

Strips	Chemical Indicator using Browne’s tubes	Autoclave tape
No Growth	Reached to 121  degree centigrade , there was change of colour from maroon to green	Tape turned black

Table 2 showing  results of sterilization of load in an autoclave using chemical and biological  indicator.

Objective of the Experiment

The strips have shown no growth due to the following reasons :

As explained earlier  B. stearothermophilus spores are known for maximum resistivity to steam sterilisation  with  at a temperature range of 50- 60 degree centigrade but they  not will survive more than 5 minutes at  autoclave temperature of 121oC. After autoclaving is complete , the  strips are  hygienically  transferred to a spore strip broth, then  incubated at 55oC.  There is a growth as turbidity of the spore strip broth increases and there is   a colour change of the ampoules. Since there is no growth  the conclusion is  that all the spore are  killed and the sterilization is successful in that part of autoclave. 

Browne’s tube contains a  heat sensitive  chemical indicator the colour of which turns from maroon red to green at a temperature of 121 degree centigrade.

Autoclave tape indicators contain colour indicator which turns back when come in contact with steam. Since it does not certify that the required temperature is reached we can not conclude that organisms are actually killed.

Autoclave uses steam held at elevated temperature and pressure for a certain time . The moist heat condenses on a surface and releases energy which split opens cell wall. The heat is able to denature proteins , effectively killing all cells present.

Conclusion :

The experiment on validation of sterilization of a load in an autoclave was conducted as per the lab manual. The learning point was the use of autoclave in sterilization process. The experiment uses spore strips using B Stearothermophilus spores, chemical indicator Browne’s tube and autoclave tape to find out  the effectiveness of the functioning of the autoclave. Hence we can say that the test results are positive. 

To find out  velocity of air across a laminar air flow cabinet,  placed inside a microbiology lab.

For effective functioning of HEPA filter a part of  a laminar air flow cabine,t the air velocity  in the  vicinity of the filter should as  low asf 0.4 meter/sec. If the velocity of air is more the no of particles can not be controlled within the desired level. So it is mandatory to have a system in place where the velocity of air near the filter should be measured from time to time.  

Using an anemometer which can measure velocity using digital counters ,the objective is  to measure the  air velocity at  the downstream side of a HEPA filter placed inside the LAF  cabinet.

Results

To maintain clean environment, laminar air flow cabinets (LAF cabinets) are used. It is highly effective to prevent the microbial cultures, cell cultures, laboratory media, microbiology labs and commercial products .from contamination.  (Whitfield, W.J.,1962,529)..

The airflow in the   LAF cabinet is unidirectional ( laminar) . The speed of air is very less, stable and parallel. LAF units are designed for  horizontal that is from the back side of the cabinet to the front side  or in a vertical way  that is from top to bottom. This experiment is conducted using vertical air flow cabinet. LAF cabinet uses (high efficiency particulate air (HEPA) filter  to block t suspended air particles. The efficiency of  removal of particle depends  on the velocity of  air while  passing through the filter. That is reason the velocity of air at the face of the filter should be measured regularly to see that the HEPA filter is working properly. The velocity is measured at selected areas in the vicinity of HEPA  filters using anemometer having digital counter.  

The specification for HEPA filter for  vertical  laminar air flow unit is of the range of 0.30 m/s –  0.40 m/s. and  for horizontal flow is  0.40 m/s –  0.50 m/s.

Lab Manual is referred.

Methods

Lab Manual is referred

Table 3: Air velocity at different zones in a laminar air flow cabinet

A. PLAN Zone             Velocity m/s	B. ELEVATION Zone            velocity m/s
1	0.52	5	0.26
2	0.37	6	0.28
3	0.31	7	0.31
4	0.40	8	0.29

The results available from the experiment clearly indicates that for both plan and elevation for all the zones the speed of air is within the accepted limit as already furnished. It indicates that HEPA filter will effectively filter air particles as the theory suggests.

Laminar air flow is defined as  flow  of air in which  air within a specified space is uniform in both velocity and direction. According to CDC the principle of laminar air plow was developed in early 1960s  still the concept is in place to maintain safe air in modern laboratories.

The results indicate that the air velocity across the laminar air flow cabinet is as per the specification that is for vertical air flow unit the speed of air is 0.35 m/s +/-0.05 except for some minor variation at zone 1, 5 and 6. The nominal air speed at the filter surface ensures  that there is a continuous change of air in the enclosed area and ensures  purity of air.

Conclusion :  The experiment on measuring  of  velocity of air across a laminar air flow cabinet , an important equipment for maintenance of clean and sterile environment in microbiology lab  was conducted as per the lab manual.  The result shows that the air velocity is consistent with the expected result. The experiment was successful. The learning point was know  about air flow cabinet and how and where to measure velocity of air using digital anemometer. 

Discussion

Determination of distribution of size of particle in a laminar air flow unit

To measure the size of particles of sizes of 0.5 micron and 5 micron in Laminar air flow cabinet using particle counter under manned and unmanned environment.

It is required to monitor regularly the level of  microbiological contamination  inside a laboratory. The number of particles of a specified   size (  0.5 m and 5.0 m  diameter)  in unit volume of air is a measure of level due to  contamination in a class room. Access to cleanrooms is generally restricted  using  airlocks( pressurized)  with minimum  two  interlock doors.. The combination of positive pressure and   air filtration using HEPA filters bring down the   level of number of particles  to abnormally low which forms the basis of cleanroom technology. The level of contamination in air is measuring  the number of particles of a given particle size  in a cm  (m³) of air at any given time. Contamination may be due to  particles contaminated by  people, equipment, microorganisms, static charge (due to rubbing of synthetic materials), chemical residues from human body, dress materials, cleaning and disinfectants.

The laser operated  counter  has three  main features : a display menu for adjusting   parameters or limits of particle count, the  serial i-o menu for adjusting  parameters on  computer interface, the  clock menu  to set time limit and hold time.

The Lab Manual is referred.

Methods

The Lab Manual is referred.

Results

TABLE 4: Record of particle count  in both  manned and unmanned environment.

Unmanned	0.5 micron	No of particles = 2
unmanned	5 micron	No of particles = 0
Manned	0.5 micron	No of particles=8
Manned	5 micron	No of particles=0

Discussion :

Particle count specification  used for inspection of clean rooms of pharmaceutical units is as per table 5.

Table 5 : Particle count specifications (from PIC/S guidelines)

	Without activity	With activity
Grade	Maximum permissible  no  of particles / m3
0.5μm	5.0μm	5.0μm	0.5μm	5 μm
A	3.5 * 103	1	3.5 * 103	1
B	3.5 * 103	1	3.5 * 105	2 k
C	3.5 * 105	2 k	3.5 * 106	20 k
D	3.5 * 106	20 k	—-	—–

Grade A: Zone  meant for high risk operations,

By the help of unidirectional air flow systems using velocity  range   0.35– 0.50 m/s. Access to  grade A area by operations people should be restricted and controlled by design.

Grade B, this area is used as background area for grade. Grade C clean rooms can interface with Grade B area.

Grade C and D area:  Less critical activities like manufacturing of sterilized  products.

This result shows that for both unmanned and manned environment the number of particles of both the sizes are very low compared to the standards given which suggests that the areas are highly sterilized.

Conclusion : The experiment  on distribution  of particle size in a laminar air flow unit controlled lab  was conducted as per the lab manual. The learning point is one of the practical uses of laminar flow cabinet sterilizing the environment using HEPA filter. The particle count gives the level of contamination of air flow. The results were in conformity with the theory and adopted in the lab manual. The experiment was a success. 

Conclusion

Pharmaceutical products are labeled sterile or non sterile based on degree of sterilization. This  practical  will use a simulated non-sterile pharmaceutical liquid. Non-sterile pharmaceutical products are   “microbiologically controlled”  products, containing a number of harmless micro-organisms at very  low level. Tablets for oral consumption  falls under this category .Only some microorganisms and very low level non-pathogenic  micro organisms are  allowed in these products, no pathogens are allowed. Testing of products are done at different stages of production to see that the process is under controlled environment  and that the product  as per the regulations at each stage (Whitfield, W.J., 1962,.529).

The main purpose is to sample the pharmaceutical manufacturing environment as a team of employees working together. So the purpose is to find out

• Contaminations from various sources.
• microbial contamination of  pharmaceutical product

through air monitoring , personnel monitoring and surface monitoring. Air monitoring is using settle plate method  and centrifugal air sampler, the personnel monitoring is through finger dab test and contact plates and the surface monitoring is through contact plates and swab. 

Monitoring of environmental parameters

(A) AIR MONITORING – Lab Manual is referred

. (B) PERSONNEL MONITORING – Lab Manual is referred

Settle Plate Method.

Results – Negative, Control- No Growth, Sample- 7 colonies round in shape. Different range in size some are small some are big milky white in colour.

Centrifugal Air Sampler

Results – Negative , Control- No Growth, Sample – 36 colonies , creamy white and circle in shape .

• PERSONELL MONIRORING

 Results – Negative,  Control- No Growth,  Sample – 15 colonies , creamy thick in size  is due to microorganism on skin .

• SURFACE MONITORING

Results – Negative  ,   Control- No Growth, Sample – 9 Colonies .

Swab

Results – Negative, , Control- No Growth, Sample – 2 colonies

Discussion  : The purpose of the practical session was to sample the extent of actual contamination of environment of pharmaceutical manufacturing site as well as to microbial contamination of a pharmaceutical products. All the tests are coming under environment monitoring. The tests are grouped as a. Air Monitoring b. Personnel Monitoring and C. Surface Monitoring.

Air monitoring was done through two tests a. Settle plate method   b. Centrifugal air sampler. Both the tests have shown negative results. There was no growth of control. Colonies developed.

In case of personnel monitoring finger dab test was conducted and the result was negative. In case of surface monitoring also the result was negative. This suggests that environment is monitored properly.

Conclusion :  The experiment on environment monitoring. was conducted as per the lab manual. The conditions stipulated and the precautions to be taken in handling different media has been followed. The learning point was to sample the pharmaceutical manufacturing environment and investigate various sources of contamination. The contamination may be due to human presence , materials used and other external factors like ineffective functioning of the equipment used for sterilization. The result was successful and in conformity with the expected results.   

Validate Sterilization of a Load by Using Biological and Chemical Indicators

Materials : Lab manual is referred

Methods

Lab Manual is referred

RESULTS :

Table 6  Sample wise result

Description of plate	Result
‘10-1’	‘Sample 1’	+ +
‘10-1’	‘Sample 2’	+
‘10-2’	‘Sample 1’	—–
‘10-2’	‘Sample 2’	—–
‘10-3’	‘Sample 1’	—–
‘10-3’	‘Sample 2’	—–
Positive  control	+++
Negative  control	—–

Symbols used in this table are

—–   means ‘No Growth’

+       means ‘some growth’

++     means  ‘ confluent growth’

+++   means   ‘Plenty of growth (TNTC)’

The colony forming unit (CFU) is generally used to measure  viable bacterial or fungal cells. In direct microscopic counts (cell count using haemocytometer)  all cells whether  dead  or  living are counted, but CFU measures viable cells only. The formula for CFU count is  CFU/ml = (No of colonies * dilution factor) / volume of culture plate.

When we want to know the number of microorganisms in a solution of fungi or bacteria, it’s usually too laborious  to count  individually each  cell using  microscope. If we  dilute  a sample of microbes and spread  it across a plate, we can  count groups of microbes, called colonies, by  naked eye. We can assume that each colony to grow  from a single colony-forming unit, or CFU. CFU count can be used to determine how many microbes were in the original sample.

Because dilution affects  CFU , at  dilution (10^-1) that means the culture is diluted 10 times. There is growth in both sample 1 and sample 2. Sample 1 has more growth than sample 2. With more dilution there is no growth. In case of test tube marked as negative control there is no growth whereas that with test tube marked positive control  there is substantial growth. .

Conclusion : The experiment was conducted as per the lab manual. The conditions stipulated and the precautions to be taken in handling different media has been followed. The results were in conformity with the theory and adopted in the lab manual. 

Standard analysis to find out  presence of coliforms in water is done  through  a number of stages: (Mates, A. and Shaffer, M., 1989, 343-346)

 The presumptive test The presence of organisms that ferment lactose by producing acid and gas is examined. The   results from a series of lactose broth tubes determine most probable number of coliform organisms per 100 ml of  water sample. Most probable number tables are available for this calculation. Further tests which are sometimes carried out include the confirmatory test and the completed tests.

The confirmatory test: The organisms that  ferment lactose (produce gas and acid) and provide  positive  test result for coliforms, are inoculated into tubes oft green bile salt broth and peptone water  confirming  the presence of coliforms.

The completed  test A final check of  colonies that develop at the time of  the confirmatory test can also be carried out. E.g.  IMViC test..

Lab Manual is referred

1. Sample -1 No Growth  is observed
2. Sample -2 No Growth is observed.

Positive control using Reverse Osmosis water – Small brown slimy colonies formed, has been inoculated.

Negative Control using Sterile Water – There is no growth.

Membrane filters are composed of substances like cellulose esters or plastic polymers. They are now-a-days  used extensively both for laboratory and industrial uses. The filters are having thickness of 0.1 mm. The pores of membrane filters include 0.22 µm and 0.45 µm sizes meant for blocking bacteria .Some flexible bacteria like spirochetes can pass through such filters. Now a days filters with pores as small as 0.01 µm is available which can block viruses.

The results of this experiment are in expected line with negative control containing sterile water has not developed any colony whereas in case of positive control using R O water small brown slimy colonies has been inoculated.

Conclusion  

Care must be taken to see that water samples are collected in sterile bottles. If water sample contains chlorine , the sample has to be neutralized using sodium thiosulphate. The samples are to be delivered to the laboratory within 6 hours of sampling. Where negative results are coming , we can expect sometimes positive results by performing confirmatory test and completed tests. The experiment on microbial analysis of water quality using various sources of water like RO water, sterile water  was conducted as per the lab manual. The conditions stipulated and the precautions to be taken in handling different media has been followed. The results were in conformity with the theory and adopted in the lab manual. 

The coagulase test is used to differentiate  pathogenic   (S. aureus ) with non-pathogenic (S. epidermidis) strains of Staphylococcus  Bacteria that produce the enzyme coagulase hence  termed ‘coagulase-positive organisms’ uses it as a   mechanism for defense  by coagulating plasma areas  around  them  which  enable themselves to resist phagocytosis by  the host’s immune system. Thus , we can conclude that most coagulase-positive organisms are pathogenic.

Two methods are in place  to carry out  coagulase test: : the tube test method  and the slide test method

. The tube test method  is more accurate, but the slide test is fasterr. The tube test method does  inoculation of  plasma of rabbit with the suspect in test tubes. If the suspect is  positive to coagulase, the  plasma  of rabbit will clot after the allotted time.

The slide method does mixing the suspect with reagents on a slide and look for clotting.  Slide test method is done in this practical ( Kateete, D.P., Kimani, C.N.,2010,23)..

To differentiate gram positive bacteria with gram negative bacteria using gram staining procedure.

The gram staining procedure is based on difference in bacterial cell wall’s structure and composition. Gram positive bacteria possess a thick peptidoglycan layer whereas in gram negative bacteria the peptidoglycan layer is very thin and surrounded by outer lipopolysaccharide layer. (Beveridge, T.J.,1999., .4725-4733).

Gram staining procedure uses four man reagents

1. Primary Stain example crystal violet that stain all cells purple.
2. Mordant example iodine that increases cell’s affinity for the stain. It binds crystal violet tightly to peptidoglycan layer thus intensify the colour of the stain.
3. Decolourising Agent example Ethanol that dissolves the outer lipopolysaccharide layer of gram negative bacteria leaving gram negative cell colourless or unstained.
4. Counter Stain example Safranin that stain decolourised gram negative bacteria pink while gram positive bacteria retains purple colour of primary stain and can be distinguished from gram positive bacteria.

For staining slides of  B Cereus, S. epidermidis, E.coli, and a mixture of  E.coli and S. epidermidis. gram staining technique is used  The logic   is that  B. cereus, and S. epidermidis  stain crystal violet color establishing  that it has a Gram positive cell wall structure and that E. Coli  is stained with the safranin pink color because of  gram negative cell wall structure.

Materials  : Lab Manual is referred

Method :  Lab Manual is referred

Results : :Observing each slide under microscope , it is  concluded that the staining is  successful. E Coli and B cereus turned deep purple from the crystal violet stain. S epidermidis turned deep pink from counter stain safranin.

After preforming gram-staining technique on the given organisms, it was observed  that  the result of this experiment matched the results of previous gram staining results.  While observing E. coli under the microscope it was concluded that the the appearance of deep pink colour indicated that it was a gram negative bacteria, therefore due to presence of  a thin layer of peptidoglycan  the crystal violet was easily decolorized out of the cell  wall by the application of  alcohol and subsequent counterstaining by safranin resulted in a pink stained cell. When observing B. cereus deep purple stain was observed which inferred that it  was a gram positive bacteria, containing a thick peptidoglycan layer that retained the crystal violet stain complexed with mordant even after subsequent washing with alcohol. This made the bacterial cells appear purple stained. When the mixture of E. Coli and S. epidermidis was observed in the slide, some purple cells and some pink cells were observed. Purple cells denoted S. epidermis and pink cells denoted E. coli in the sample containing mixture of a gram positive and gram negative bacteria. Since E.coli is already found to be gram negative, purple coloured gram positive cells in the mixture must be S. epidermis. This was further confirmed when observing the slide containing calls of only S. epidermidis, only purple coloured cells were observed in it indicating that it was a gram positive bacteria having a thick peptidoglycan layer in its cell wall.

Conclusion :  The experiment on coagulase test using gram stain procedure was conducted as per the lab manual. The conditions stipulated and the precautions to be taken in handling different media has been followed. The results were in conformity with the theory and adopted in the lab manual. 

References:

Tripathi, K.D., 2013. Essentials of medical pharmacology. JP Medical Ltd.

David, J.R., Graves, R.H. and Szemplenski, T., 2012. Handbook of aseptic processing and packaging (Vol. 4). Crc Press.

Beveridge, T.J., 1999. Structures of gram-negative cell walls and their derived membrane vesicles. Journal of bacteriology, 181(16), pp.4725-4733.

Kateete, D.P., Kimani, C.N., Katabazi, F.A., Okeng, A., Okee, M.S., Nanteza, A., Joloba, M.L. and Najjuka, F.C., 2010. Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test. Annals of clinical microbiology and antimicrobials, 9(1), p.23.

Mates, A. and Shaffer, M., 1989. Membrane filtration differentiation of E. coli from coliforms in the examination of water. Journal of applied bacteriology, 67(3), pp.343-346.

Whitfield, W.J., 1962. A New Approach to Clean Room Design: Annual Proceedings. In Institute for Environmental Sciences(p. 529).