Aseptic Techniques (Microbiology report)

ASEPTIC TECHNIQUES AND SOURCES OF MICROBIAL CONTAMINATION.

Introduction

The spread of infections has come to a point where it has become catastrophic. Aseptic technique is the method used to prevent contamination of infections. It is widely used in hospitals, pharmacy, and pharmaceutical industries and in laboratories. Different establishments have come up with more ways to improve infection control.

In hospitals health care acquired infections are costing the NHS £1 Billion a year and have been causing 5000 deaths annually. Aseptic non-touch technique (ANTT) has been advocated as a standardized procedure to all U.K hospitals. i

Sources of infections are healthcare professionals who could be sick themselves or even carriers of infection and yet are not aware of it. To prevent spread of infections, the members of staff are usually given liberal sick leave, screened for Hepatitis, TB, staphilococus aureus to name but a few. Infection control also helps protect the health care professionals from being contaminated by patient’s bodily fluids, blood and toxic substances.

The hospital environment could also be a source of infection hence measures have been taken to try and reduce the risks by regular disinfection and sterilization, providing good ventilation to the rooms, designed infrastructures that cater for areas where strict aseptic procedures and strict protocols are followed such as washing and disinfecting hands after each patient contact to prevent cross contamination from one patient to another, infected patients are isolated, single use of equipment and proper sterilization of non disposable equipment.

Microbial contamination could cause physiochemical changes of the medicines. This could alter the contents of the active ingredients or convert them into toxic products.ii

In research laboratories where cell cultures are grown there is a high risk of contamination by micro-organisms the sources here could be the cells used in cultures, the glassware or apparatus although they have been autoclaved; improper maintenance or sterilization of autoclaves may cause serious contamination out breaks in industries at large. As Stacey (2011) mentions that attention to training of staff, laboratory layout, appropriate use of quarantines for new cultures or cell lines, cleaning and maintenance and quality control are important in preventing contamination in cell culture laboratories.iii

Aim
In this experiment we investigate the sources of contamination in the laboratory and how aseptic techniques and correct handling of microorganisms can help infection control.

Method: Please refer to the module handbook.

Results

Activity A: Correct handling of Micro-organisms (Aseptic Technique)

Table 1: Growth on plates

Types of cultures streaked on to
Nutrient Agar

Escherichia coli
Serratia
Amount of growth
Sectors 0-1
+++
Sectors 0-1
+++

Sectors 1-2
+++
Sectors 1-2
+++

Sectors 3
±
Sector 3
+

Sectors 4
0
Sector 4
±
Were single colonies obtained?

Yes

Yes

Number of colony types

1

1

Key

0 No Growth
± Occasional colonies
+ Light Growth
++ Moderate Growth
+++ Heavy Growth

Table 1. Shows the growth of cultures of the nutrient agar plate that was streaked with E.coli. The plate produced growth as follows; sectors 0-1 heavy growth, sectors 1-2 there appeared to be moderate growth of colonies, sectors 3 appeared to have occasional colonies produced while sector 4 had no growth at all.
Also from the table it can be seen there was culture growth on the nutrient agar plate that was streaked with Serratia. The results for this were as follows; sector 0-1 produced heavy growth, sector 0-2 appeared to have a heavy growth of colonies, sector 3 had a light growth and sector 4 appeared to have occasional colonies.

Table 2: Growth in broth cultures

Broth cultures
Growth in broth cultures
Sterile nutrient broth A
A
0
Loop transfer of sterile broth A to sterile broth B
B
+
Inoculation of sterile broth with Escherichia coli. By loop transfer.
C
++

Key

0 No growth ( broth as clear as uninoculated broth).
+ Slight visible growth.
++ Moderate growth- distinct turbidity but you can still see through the tube.
+++ Heavy growth- the tube is so turbid so you can not see through it.

Table 2 Shows the results for the loop transfer of sterile broth (A) to sterile broth (B) and the inoculation of sterile broth (A) with bacteria (Escherichia coli) on nutrient agar plates, broth culture C.
Here it can be seen that the sterile nutrient broth A had no growth of cultures at all. Loop transfer of sterile broth A to sterile broth B, had a slight visible growth and broth culture C which was the inoculation of sterile broth A with E.coli by loop transfer had a heavy growth.

Source of Contamination

Type of medium
(Agar)

Air
(MCP/30min)
Surface of bench before sanitizing
(CFU/100cm2)

Surface of bench after sanitizing
(CFU/100cm2)

Human Body (Female)
(1.7 m2)

Nutrient

31

804

0

5.7 x105

Sabouraud

0

0

0

0

Activity B : Investigation of sources of microbial contamination.

Table 3: The number of colonies on each plate:

From Table 3. It can be observed that the nutrient agar in all the different conditions produced growth of colonies. However, it can be noted that the Sabouraund agar did not have growth of colonies at all.

From the Air, Nutrient agar had a growth of 31MCP/30min while the human body produced 5.7 x 105 colonies per human body with a surface area of approximately 1.7 m2.

The surface of the bench before being sanitized had a growth of 804 CFU/100cm2 . On the contrary, the sanitized bench did not have any colony forming units.

Calculations:

Microbes detected on the surface of bench before sanitization:

Area investigated =25cm2
Colonies growth on nutrient agar, > 200
If 25cm2 contained 201 colonies.
Therefore, 804CFU/100cm2

Microbes detected from the air

Colonies obtained in 30minutes from air = 31 microbe carrying particles /30min

Microbes detected from the surface of human body

Surface area of fingertips used to contaminate agar is 3cm2. Average surface area of average female is approximately 1.7m2.
Colonies detected from 0.0003m2 was 101.
Therefore, colonies per human body will approximately be

(101x1.7)÷0.0003m2

= 5.7x105

Discussion

Activity A was an experiment on the correct handling of micro-organism. Here, we see when correct aseptic technique procedures are used you may reduce contamination to a very high level. In this activity Streaking is used to identify and isolate pure bacterial colonies from a mixed population. Microbiologists use bacterial and other microbial culture streaking methods to identify microorganisms and to diagnose infection.iv When the nutrient agar was streaked with E.coli by loop transfer aseptically, the aim was to produce single colonies of a pure culture of the E.coli. Because the aseptic technique was done correctly, there was no contamination found on the plates also single colonies were formed. This was the same for the streaking of the serratia. Single colonies were obtained from the streaking the plate aseptically and there was completely no contamination at all.

Table B shows the sterile nutrient broth A had no growth of cultures at all, this is because broth A was not contaminated with anything else and the loop transfer was done aseptically. The loop transfer of sterile broth A to sterile broth B, had a slight visible growth this is because sterile broth B was contaminated with sterile broth A, hence the plate contained 2 broths. The broth culture C was inoculated broth culture A with E.coli. This is where there was heavy growth because bacteria were introduced; there was complete microbial contamination.

Activity B investigates the sources of microbial contamination. Table 3 shows that the Sabouraund agar (SAB) had no growth at all on all the different conditions. This is because it contains mycological peptone and relatively high concentrations of sugar, it has a pH of 5.0 and it inhibits growth of many bacteria however it supports of many yeast and fungi.v
The nutrient agar (NA) on the other hand, supports the growth of many non-fastidious bacterial species this is why it produced growth of cultures. In the laboratory Air, nutrient agar had a growth of 31 microbes carrying particles/ 30 minutes. This is the amount of colonies that were formed on the media when it was left in open air for half an hour. This shows that in the air, there are lots of microbes. Especially in a laboratory or hospital environment where there could be air borne infections spreading from one person to the next. In hospital environment, there is plenty of ventilation supplied into the wards to reduce this risk. Patients who are infected may cause a risk to other patients are usually kept isolated, to reduce the spread of infections.
The surface of the bench was swabbed before and after sanitizing. Before sanitizing it was found out to have been carrying 804 CFU/100cm2 however, after it was sanitized using 70% iso-propanol the bench did not have any microbes at all. 70% Iso- propanol is a standard disinfection that is used in pharmaceutical manufacture. It can be seen that correctly sanitizing surfaces does really help reduce the spread of microbes. In hospitals or laboratories, microbes are spread everywhere and tend to land on surfaces of tables, bead steads, door handles, window seals etc. However, if correct sanitization is done routinely this risk of cross contamination of infections could be highly reduced.
The human body is another source of microbial contamination especially the hands. Table 3 shows that the human body produced 5.7 x 105 colonies per human body with a surface area of approximately 1.7 m2. It can be seen from this it is clearly seen that our bodies could be carriers of micro- organisms and yet we don’t even know it. This is why hand washing , and sterilisation is highly emphasized to maintain an overall good hygiene. Hands go everywhere, touching door handles where an infected person may have touched, people cover their mouths when coughing hence all the microbes from the respiratory tract can be transferred into the hands and then spread by either hand shaking or just simply answering a phone call in the office! The next person answering the same phone can get all the microbes from the first person that used the phone. This is why in hospitals, health care professionals are trained on the ANTT (aseptic non touch technique) and also correct washing of hands before and after each patient contact as this reduces cross contamination.vi

In conclusion, when aseptic technique procedures are carried out routinely in hospitals, laboratories or pharmaceutical industries. There will be a reduced risk of microbial contamination. However, staff should be fully trained and updated on new methods of increasing asepsis. Awareness, of aseptic technique and importance of hygiene if made to the public (in the community) will also help reduce infectious diseases for examples having posters placed in public toilets showing the proper hand wash procedure and reminding people of importance of washing their hands after a visit to the toilet.