Isolation And Identification Of Air Microflora In Microbiology Laboratory

Project and Seminar Material for Microbiology

Project and Seminar Material for Microbiology


Microflora contamination in laboratories and hospitals is becoming a serious problem worldwide, and the characterization of such contaminants offers hope for the treatment of some infections acquired in hospitals and laboratories (LAI). Microflora contamination in benches, floors, media and equipment can be affected by temperature, humidity, nutrient media in laboratories and media storage conditions. Therefore, microflora sources must be determined, contaminants must be isolated and identified when standard microbiological manipulations are performed.

The objectives of this study were

  1. To determine the sources of microflora contaminants in microbiological laboratories in Nigieria
  2. To identify bacterial and fungal contaminants in biosafety laboratories selected based on morphological and biochemical properties and
  3. Genetic determination Identity of persistent Nigieria bacteria in laboratory sites after disinfection with sodium hypochlorite. The isolation of pure cultures was performed on the basis of morphological differences, using the shape of the colony, elevation, pigmentation and size to distinguish bacterial and fungal contaminants.

The results showed that

  1. The laboratory sites examined were contaminated with different microbes.
  2. Macroscopic and microscopic observations of fungi confirmed the presence of Cladosporium sp, Penicillium sp, Aspergillus sp and Alternaria sp on tables, door handles, preparation rooms, gloves and biosafety cabinets, and
  3. The persistent bacteria identified were Shigella sp., Pseudomonas aeruginosa, Corynebacteria sp., Bacillus sp. and Staphylococci aureus.

The contaminants were similar to the standard strains, but there was a significant difference in contamination in the three selected laboratories (analysis of variance (ANOVA P = 0.00)). The size of the PCR product was 996 bp and the RFLP patterns of the bacteria were concluded that despite the disinfection with sodium hypochlorite, bacterial and fungal contaminants remain on laboratory surfaces and equipment and, therefore, they should increase the concentration or change the disinfectant.

Chapter One


Gases, dust particles, water vapour and air contain microorganisms. There are vegetable cells and spores of bacteria, fungi and algae, viruses and protozoa cysts. Since air is often exposed to sunlight, it has a higher temperature and less moisture. So, if not protected from desiccation. Most of these microbial forms will die. Air serves as transport or dispersal medium for microorganism they occur in relatively small number in air when compared with soil or water. The microflora of air can be studied under two headings outdoor and indoor microflora.

Outdoor Microflora:

The air in the atmosphere, which is found outside the buildings, is referred to as outside air. The dominant microflora of outside air are fungi. The two common genera of fungi are cladosporiul and sporobolomyces, besides this two general, under general found in air are Aspergillus, Alternaria, Phytophthora and Erysiphe. The outdoor air also contains besidispores, ascopres of yeast, fragments of mycelium and canidia of molds. Among the bacterial genera Bacillus and clostridium, sarcina, mirococcus, corynebacterium and Achromobacter are widely found in the outside air, the number and kind of microorganism may very from place to place, depending upon the human population densities.

Indoor Microflora:

The air found inside the building is referred to as indoor air. The commonest genera of fungi in indoor air are penicillium, Aspergillus, the Commonest genera of bacteria found in indoor air are Staphylococci, Bacillus and Clostridium. In case of occupants being infected, the composition shows slight variations with latitude and to a lesser extent with attitude. The ozone owes its existence in the atmosphere to photosynthesis from oxygen under the influence of solar ultraviolet radiations. (Dr. Shiva, 2009).

There is no microbes are native to the atmosphere rather they represent allochthonous populations transperted from aquatic and terrestrial habits into the atmosphere. Microbe of air within 300 – 1,000 or more feet of the Earth’s surface are the organisms of soil that have become attached to fragments of dried leaves, strain or dust particles, being blown away by the wind. Species vary greatly in their sensitivity to a given value of relative humidity, temperature and radiation exposure.

More microbes are found in air over land masses than far at sea. Spores of fungi especially Alterneria, Cladosporium, Penicillium and Aspergillus are more numberous than other forms over sea within about 400 miles of land in both polar and tropical air masses at all altitudes up to about 10,000 feet.

Microbes found in air over populated land areas below altitude of 500 feet in clear weather include spores of Becillus and Clostridium ascos-pores of yeasts, fragments of mycelium and spores of molds and streptomycetaceae, pollen pootozoan cysts, algae, micrococcus, corynebacterium etc.

In the dust and air of schools and hospital wards or the rooms of persons suffering from infectious disease, microbe such as tubercle bacilli, streptococci, pneumococci and staphylocci have been demonstrated. These respiratory bacteria are dispersed in air in the droplets of saliva and mucus produced by coughing, sneezing, talking and laughing.

Viruses of respiratory tract and some enteric are also transmitted from the objects contaminated with infectious secretions that after drying become infectious dust. Droplet are usually formed by sneezing, coughing and talking. Each droplet consists of saliva and mucus and each may contain thousands of microbes. It has been estimated that the number of bacteria in a single sneeze may be between 10,000 and 100,000. Small droplets in a warm dry atmosphere are dry before they reach the floor and thus quickly become droplet nuclei.

Many plant pathogens are also transported from one field to another through air and the spread of many fungal diseases of plants can be predicted by measuring the concentration of airborne fungal spores. Human bacterial pathogen which cause important airborne disease such as diphtheria, meningitis, pneumonia, tuberculosis, and whooping cough are described in the chapter “Bacterial Disease man”

Air does not have an indigenous and flora, though a number of micro-organism are present in the air. Air is not a natural environment for microorganisms as it doesn’t contain enough moisture and nutrients to support their growth and reproduction. Quite a number of sources have been studied in this connection and almost all of them have been found to be responsible for the air microflora. One of the most common sources of air microflora is the soil. Soil microorganisms when distributed by the wind blow librated into the air and remain suspended therefore along period of time. Man made actions like digging or ploughing the soil may be release soil born microbes into the air. Similarly microorganisms found in water may also be released into the air in the form of water droplets or aerosols, splashing of water by wind action a tidal action may also produce droplets or aerosols?

1.2 Problem statement

Microflora contamination in laboratories and hospitals is becoming serious problem worldwide and characterization of such contaminants offer hope for treating some laboratory acquired infections (LAI). It is important to determine the nature of micro-organisms which are as result of such accidents inorder to devise mitigation techniques before outbreaks (Porte et al., 2012). To exploit the benefits associated with cell culture procedures, there are laboratory quality and biosafety practices that are overlooked, that lead to microflora contamination in large numbers in the cell cultures and in laboratory environment which may lead to (LAI) to people (Jizhou et al., 2014). The contamination risks pose danger to laboratory personnel as well increase the costs of in-house cell culture procedures (Niimi et al., 2011).

1.3 Objectives of the study

To isolate, identify and characterize bacteria and fungi contaminating selected microbiology laboratories in Nigeria. Specifically we aim:

  1. To determine sources of microflora contaminants in selected microflora laboratories in Nigeria.
  2. To isolate and identify the bacterial and fungal contaminants in microbiology laboratories based on morphological and biochemical characteristics.
  3. To evaluate the genetic identity of persistent bacteria in laboratories after disinfection with sodium hypochlorite.

1.4 Significance of the study

The information from this study will help to contain the Laboratory Acquired Infections) LAI associated with microbial contaminations in microbiology laboratories. The research findings will also assist personnel in laboratories to be careful when performing standard manipulations of microbiological specimens in cell cultures.

This will help in reducing the costs associated with the application of the technology in laboratories. The information will form a basis of training the personnel on capacity development in monitoring and evaluation of microflora contaminants in laboratories. Then effecting or upgrading policy on Standard Operating Procedures (SOPs) will be developed in the laboratories.

1.5 Research questions

  1. What are the sources of microflora contaminants in microflora laboratories in Nigeria?
  2. Are the bacterial and fungal contaminants encountered in microbiology laboratories different morphologically?
  3. Are the persistent bacteria to hypochlorite based disinfection genotypically similar to standard bacteria strains?

Chapter Five

Conclusions And Recommendations

5.1 Conclusions

  1. 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.
  2. Each site contained more than one microflora contaminants.
  3. In objective two, the isolated bacteria and fungi contaminants are similar based in morphological and microchemical characteristics among the laboratories.
  4. The bacteria persistent to disinfection isolated were Pseudomonas sp, Staphylococcus aureus, B. subtilis, Corynebacterium sp and Shigella sp.
  5. 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.

5.2 Recommendations

  1. 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.
  2. 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.
  3. Universal PCR followed by RFLP could be considered as a simple and sensitive method for detection and identification of bacteria.
  4. 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.
  5. 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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