Isolation and Identification of Air Microflora in Microbiology Laboratory
Chapter One
Objectives of the study
To isolate, identify and characterize bacteria and fungi contaminating selected microbiology laboratories in Nigeria. Specifically we aim:
- To determine sources of microflora contaminants in selected microflora laboratories in Nigeria.
- To isolate and identify the bacterial and fungal contaminants in microbiology laboratories based on morphological and biochemical characteristics.
- To evaluate the genetic identity of persistent bacteria in laboratories after disinfection with sodium hypochlorite.
CHAPTER TWO
LITERATURE REVIEW
Major cell culture contaminants in biosafety laboratories
A cell culture contaminant can be defined as some element in the culture system that is undesirable because of its possible adverse effects on either the system or its use (Wolfang, 2008). These elements are caused by biological contaminants.
Causes of biological contamination
Biological contamination is best described as the presence of living substance that results in undesirable effects on the culture system (Negi et al., 2011). Even essential nutrients become toxic at high concentrations. The media, water, sera, storage vessels and fluorescent light causes biological contamination (Lane et al., 1985).
Media
Majority of the biological contaminants are found in cell culture media and come either from the reagents used to make them or the additives such as sera used to supplement them. Reagents should always be of the highest quality and purity and must be properly stored to prevent deterioration. Ideally, they should be either certified for cell culture use by their manufacturer or evaluated by the researcher before use. Mistakes in media preparation protocols, reading reagent bottle labels or weighing reagents are other common sources of bacterial and fungal contamination (Odutayo, 2004).
Sera
Sera used in media have long been a source of biological contaminants. Due to cell culture-based screening programs currently used by good sera manufacturers, it is unusual to find a lot of fetal bovine sera that is toxic to a majority of cell cultures. However, it is common to find substantial variations in the growth promoting abilities of different lots of sera for particular cell culture systems, especially for cultures that have specialized or differentiated characteristics (Freitas et al., 2008). Uncontrollable variation in hormone and growth factor concentrations makes this problem inevitable. Careful testing of sera before purchase or switching to serumfree media can avoid these problems.
Water
The water used for making media and washing glassware is a frequent source of contamination and requires special care to ensure its quality (Freitas et al., 2008). Traditionally, double or triple glass distillation was considered to be the best source of high quality water for cell culture media and solutions (Greisen et al., 1994). However, these systems must be properly maintained and serviced to ensure continued water quality (George, 1993). Because of its aggressive solvent characteristics, highly purified water can be a source of bacteria contaminants from glassware or metal pipes and plasticizers from plastic storage vessels or tubing. These contaminants can then end up in media or deposited on storage vessels and pipettes during washing and rinsing. Water used to generate steam in autoclaves may contain additives to reduce scale build up in pipes. These potentially toxic additives can also end up in glassware. Other sources of contaminants include storage vessels, fluorescent lights and incubators.
CHAPTER THREE
MATERIALS AND METHODS
Study design
The study was carried out at the osun state Polyethnic IREE, it was carried out in the microbiology laboratory.
Media preparation
All the media were purchased from Laboratory and Allied company, Lagos. The specific media for bacteria isolation and growth supplied in powder form was weighed, prepared and reconstituted using distilled water. The agar was made of 28 grammes (g) per litre for bacteria growth (digest of animal tissue 5.00 g, beef extract 1.50 g, yeast extract 1.50 g, sodium chloride 5.00 g, agar 15.00 g) was prepared by suspending in 1000 ml of distilled water and the pH was adjusted to 7.4 using 2M NaOH (Tokuyasu et al., 2012). The agar mixture was dissolved in water by boiling at 100 °C then the agar was sterilized by autoclaving at a pressure of 15 pascals and a temperature of 121 °C for 15 min. Nutrient broth (with the same ingredients except agar) was prepared by suspending 13.0 g of nutrient agar in 1000 ml distilled water and heated to dissolve completely then sterilized as above.
Salmonella Shigella Agar (SSA) medium (peptic digest of animal tissue 5.00 g, beef extract 5.00 g, lactose 10.00 g, bile salts mixture 8.50 g, sodium citrate 10.00 g, sodium thiosulphate 8.50 g, Ferric citrate 1.00 g, brilliant green 0.00033 g, neutral red 0.025 g, agar 15.00 g) all in one liter volume was prepared by suspending 63.0 g SSA in 1000 ml distilled water and the pH adjusted to 7.0 using glacial acetic acid. The mixture was warmed with frequent agitation to dissolve the medium completely in water. It was then cooled to a temperature of 50 °C, mixed well and poured into sterile petridishes. Mannitol Salt Agar (proteose peptone 10.00 g, beef extract 1.00 g, sodium chloride 75.00 g, mannitol 10.00 g, phenol red 0.025 g in a one litre volume) was used for selective isolation of pathogenic Staphylococcus aureas. Fifteen grams of mannitol agar was dissolved in 1000 ml of distilled water. The pH was adjusted to 7.4 using glacial acetic acid. It was boiled to dissolve the medium completely then sterilized by autoclaving at a pressure of 15 pascals and a temperature of 121 °C for 15 min.
CHAPTER FOUR
RESULTS
Isolation and identification of contaminants in biosafety laboratories
Under objective one, a total of seven bacterial isolates were obtained and identified from different laboratory places. The contaminants were the same in all the laboratories from similar sites. The bacterial contaminants were Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus, Bacillus cereus, B. subtilis and Corynebacterium sp.
CHAPTER FIVE
CONCLUSIONS AND RECOMMENDATIONS
Conclusions
– In objective one, all tested sites had microflora contaminants. The contaminated areas were in media preparation room, incubating room, laboratory walls, tables, laboratory dust coats, biosafety cabinets, door knobs and gloves used in biosafety cabinets.
– Each site contained more than one microflora contaminants.
– In objective two, the isolated bacteria and fungi contaminants are similar based in morphological and microchemical characteristics among the laboratories.
– The bacteria persistent to disinfection isolated were Pseudomonas sp, Staphylococcus aureus, B. subtilis, Corynebacterium sp and Shigella sp.
– Since there were no changes in restriction patterns of local isolates in comparison with standard bacteria, it seems that there has been no particular mutation in these local isolates. Rapid detection and identification, high sensitivity and specificity and reduced expenses are the significant advantages of this universal PCR method.
This study shows that laboratories in Osun state polyethnic are contaminated with a wide variety of potentially pathogenic bacteria and harmful fungus. Therefore the hypothesis of study is accepted. Bacterial contamination remains a continuing threat in microbiology laboratories, but techniques for reducing contamination are available.
Recommendations
– Proper working in aseptic conditions by operators and attending to the maintenance and use of autoclaves, laminar flow hoods and growth rooms are the first important steps toward avoiding environmental contaminants. Indexing cultures at the initiation stage and again throughout the culture cycle is a second step which can significantly reduce the number of contaminants that escape detection. The third step is to identify persistent possibly endophytic contaminants with antibiotics to determine correct concentrations for effective treatment and minimal phytotoxicity. This calls for thorough quality assurance enforcement and monitoring of specimen in biosafety laboratories to eliminate these contaminants.
– Molecular identification methods should be used as an adjunct to first line phenotypic identification schemes for bacteria and fungi, where a definitive identification is required. Where the use of molecular identification methods is justified, employment of partial rDNA PCR and sequencing provides a valuable and reliable method of
identification of environmental bacteria and fungi.
– Universal PCR followed by RFLP could be considered as a simple and sensitive method for detection and identification of bacteria.
– All the personnel must use dust coats which should be cleaned daily and must wear laboratory canvas once in the labs to prevent
introduction of microbes to the laboratories.
– Laboratories must assess their situation, determine contamination sources, and change their laboratory operations to avoid or eliminate most of the contaminants. Several logical steps can be taken to greatly reduce bacterial contaminants in these laboratories.
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