INTRODUCTION

The major difference between bacteria and fungi (mould/mold) in terms of biology is that bacteria belong to a diverse group of organisms called prokaryotes and fungi are eukaryotic organisms.  Prokaryotes differ from eukaryotic organisms primarily in their lack of a membrane-bound nucleus and lack of extensive endoplasmic reticula and mitochondria.  Their double-stranded DNA is found not in a nucleus (eukaryotes) but throughout the cell’s cytoplasm.  They range in size from 1-10  m.  Bacteria can be isolated using general, selective, and/or differential microbiological media incubated under the proper conditions of temperature, humidity and presence or absence of oxygen.  They are isolated in pure culture and subjected to a series of physiologic and biochemical tests to identify them to genus and species level.

A fungus is a nonmotile, nonphotosynthetic eukaryotic microorganism that contains a nucleus, produces filamentous and branched structures and has a cell wall that contains chitin or cellulose.  Fungi absorb nutrients from their environment.  Fungi and bacteria are responsible for the decomposition of dead organic material in the  environment.  Fungi exist as either a yeast form; as a filamentous form (mould/mold) or rarely as both forms dependent on the incubation temperature.  The term mould or mold is a term, usually considered a lay term for filamentous fungi.

Procedures for the identification of bacteria, yeast and filamentous fungi are different.  Bacterial identification is based upon morphological and biochemical characteristics and almost similar in case of yeast.  Morphology is used to determine the yeast to genus level and biochemical characteristics are used to determine to species level.  Filamentous fungi (moulds/molds) are identified based upon colony characteristics and microscopic characteristics.

Major Characteristics of Prokaryotes (Bacteria and Blue-Green Algae) and Eukaryotes (Fungi, Protozoa, Plants, and Animals).

CHARACTERISTIC	PROKARYOTES	EUKARYOTES
Membrane-bound organelles (including nucleus, mitochondria	Absent	Present
Cell wall, when present	Complex, contains peptidoglycan	Simple, may contain cellulose, chitin
Genetic information	Single chromosome or even sometimes represented by either of the Nuclic (DNA/RNA)	Multiple chromosomes, located in the membrane-bound nucleus
Contain both DNA and RNA	Present	Present

BACTERIA

Bacteria have rigid cell walls which are responsible for their cell shape.  Bacteria appear as either cocci or bacilli.  Bacteria that appear as cocci are spherical and occur in pairs, grape-like clusters, or chains.  Bacteria that appear as bacilli are rod-like.  The cell wall of a bacterium is responsible for the Gram-stain reaction.  The cell wall of Gram-positive organisms contain a thick peptidoglycan layer and teichoic acid.  The cell wall of Gram-negative organisms contain a single peptidoglycan layer attached to a lipopolysaccharide phospholipid bilayer outer layer (LPS).  The cell wall is responsible for protection of the bacterium from host defenses and the environment.  It may be covered by either a mucoid capsule or a slime layer, or both.  It may have finger-like projections (pili) to anchor the bacterium to its environment or it may have flagella which allow the bacterium to move.

Some bacteria are capable of producing a resting structure termed a spore.  The spore is resistant to adverse changes in their environment.  The most common change is the drying out of the environment which kills the bacterium’s vegetative cell but the spore remains  viable (Perinate) but in a resting state.  When conditions are suitable, the spore will generate a new vegetative cell.

There are two basic types of microbiological media for isolation of bacteria: routine isolation media and enriched isolation media.  The pH of both groups of media used to isolate bacteria from clinical specimens and environmental samples is between 6.8 and 7.2 with 7.0 being neutral pH.  The routine isolation media chosen should be capable of isolating the majority of the bacterial organisms that are found in the environment.  The two most commonly used media are Tryptic Soy Agar (TSA) and R2A agar.  If the suspected bacteria require special nutrients, such as Legionella species or beta-hemolytic streptococci than enriched isolation media (should be used with the routine media. Buffered charcoal yeast extract agar with  -ketoglutarate (BCYE ) incubated at 35^oC (95^oF) is capable of growing Legionella species found in environmental water samples.  Tryptic Soy Agar (TSA) containing 5-10% sheep blood incubated at 35^oC (95^oF) is capable of growing organisms found in hospital and nursing home environments, such as beta-hemolytic streptococci.  Another example is the use of gram-negative media for the isolation of gram negative bacilli from water samples.

The main isolation temperature for environmental bacteria should be 30^oC (88^oF),  not at the temperature, 35^oC (95^oF), that clinical laboratories incubate their bacterial cultures.  This is because there are important environmental bacteria that will not grow at 35^oC (95^oF)  but grow at 30^oC (88^oF).  Cultures for Legionella species and other bacterial human pathogens should be incubated at 35^oC ± 2^oC (95^oF). 

ACTINOMYCETALES

Members of the Order Actinomycetales are gram-positive bacteria that range in morphology from coryneform (bacilli) to well developed branching filaments that resemble fungal hyphae.  Members of the actinomycetes are found in greatest numbers in soil.  They have three important roles: decomposition of the organic matter found in soil; binding clay particles to their filaments which is conductive to crop production; and responsible, in part, for the earthy odor of soil.  The majority of aerobic actinomycetes are capable of growth at 25-30^oC, however, some genera are capable of growth at 50^oC (thermophiles).  Some genera are partially acid fast.  Non-thermophilic actinomycetes are commonly isolated on media used for the isolation of fungi.  Aerobic actinomycetes that are fre­quently encountered in the environmental microbiology laboratory include Strep­tomy­ces, Rhodo­coccus, Nocardia, Mycobacte­rium, Actino­madura, Nocardio­psis, and Dermat­ophil­us.  Three genera of thermophilic actinomycetes are considered to be important clinically: Saccharopolyspora species; Saccharomonospora species; and Thermoactinomyces species.  The aerobic actinomycetes are ubiquitous and can be found in water, air, soil, compost piles, home and industrial air-conditioning systems, house dust, hay and bagasse.  The pathogen­ic aerobic actinomycetes usually cause a chronic exudative inflammatory infec­tion but may occa­sionally cause an acute, necrotizing pyo­genic infection.  The repeated  inhalation of dust containing thermophilic actinomycetes or their spores may result in either hypersensitivity pneumonitis (farmer’s lung) or extrinsic allergic alveolitis ( a serious, disabling, immunologically mediated pulmonary disease that may affect agricultural, office, and industrial workers).

FUNGAL ORGANISMS

A fungus is a nonmotile, nonphotosynthetic eukaryotic microorganism that contains a nucleus, produces filamentous and branched structures and has a cell wall that contains  chitin or cellulose.  The filamentous and branched fungi elements spread over and through the substrate that the fungus will use for food.  The filamentous fungus will grow on the surface of the substrate that it is attached to.  In situations where there has been water damage, the hyphae can attach to wood, to the wallboard or to any foreign body available.  As the fungus continues to grow, specialized structures that produce spores will form.  These specialized structures can produce one spore or hundreds of spores dependent upon the species.  Each spore is capable of reproducing alike colony of fungus.  Fungi and bacteria are responsible for the decomposition of dead organic material in the environment.  Fungi that exist as a yeast grow in bacterial-like colonies and reproduce by budding. 

There are approximately 100,000 different species of fungi found in the environment.  Approximately 50-60 species are responsible for causing cutaneous, subcutaneous, and systemic infections in humans.  Fungi cause disease in humans and animals by: inhalation of spores (systemic infections); traumatic implantation into tissue  (subcutaneous infections); and infecting dead skin cells (cutaneous [ringworm]).

Mycotoxins are organic compounds produced by the fungus that can cause food poisoning caused by eating contaminated foodstuffs and grains or by eating poisonous mushrooms.  Allergic (hypersensitivity) reactions can occur following inhalation of fungal spores or mycotoxins.

The filamentous fungi can be divided into four major groups: Phycomycetes, Ascomycetes, Basidiomycetes, “Deutromycetes” or and Fungi Imperfecti.  The Zygomycetes are rapid growers and will fill a petri dish within a few days.  Cultures are usual­ly white to tan, gray to black and have a woolly texture.  Rarely, certain species can be an opportunist human pathogen causing zygomycosis, especially in diabetic patients.  The Ascomycetes are the most common of environmental filamentous fungi and are characterized by the development of asci, saclike cells that usually contain eight ascospores.  The Basidiomycetes include both poisonous and nonpoisonous mushrooms and besides rust and smut and produces basidio spores (generally 4 in number).  Deutromycetes lack sexual stage and reproduced by asexually produce spores like Conidia.  Apart from this there are a number of other fungi trapped from the ambient air does not produce spores at all.  Therefore, cannot be identified and may be grouped as Mycelia Sterilia.  In general, fungus reported from the air are collectively known as airborne fungi.

Fungi prefer an acid environment for growth, therefore, the optimum isolation medium for fungi should have a pH of 6 or less.  Many fungi can grow in media that have a pH as low as 4.5.  Malt extract agar [MEA], pH 4.7, is used as a primary isolation medium for the isolation and propagation of yeasts and filamentous fungi.  The use of a medium with a pH between 4.5 and 5.5 is capable of inhibiting the growth of bacteria because of the low pH.

The majority of fungi will grow between 0^oC (32^oF) and 35^oC (95^oF) with an optimum temperature between 25^oC (77^oF) and 30^oC (88^oF).  Incubation temperature for isolation of environmental fungi is recommended at 25^oC (77^oF).  Cultures for pathogenic fungi (human) should be incubated at 30^oC (88^oF) and 35^oC (95^oF).  Cultures for thermophilic fungi should be incubated at 50^oC (122^oF).

Fungi are capable of growing independently of bacteria and bacteria are capable of growing independently of fungi in the environment or in environmental samples.  While both have a requirement for temperature, water and nutrients, and many of the substrates that they are capable of growing on are similar, bacteria and fungi  are independent of each other.  Bacteria grow best in a neutral pH (6.8-7.2) environment while fungi prefer an acid pH (4.5-6.0) environment.

TEMPERATURE

Most bacteria and fungi have an optimum temperature at which they grow and divide most rapidly.  They also have a maximum and minimum temperature, above and below which the organisms will not grow and divide.  As you increase the temperature from the minimum temperature a 10 C increase results in a doubling of cell growth.  Above the optimum temperature the temperature gets too high and enzymes and cell structure are denatured and the cell dies.  Organisms can be grouped based on their temperature profile.

Psychrophile	opt 5-20 C
Mesophile	opt 20-50 C
Thermophile	opt 50-80 C
Extreme Thermophile	opt 80 C

Thermophiles have proteins that are not denatured because of their structure and bonding.  These groupings are not sharp divisions and organisms often cross divisions or only grow within a small range within a division i.e. some mesophiles grow at 45 C, others do not.  The temperature profile often reflects the natural growth conditions/habitat of the organisms.  Hence, the possibility of thermophilic and Chrysogenic bacteria cannot be completely ruled out into the indoor environment. 

LIGHT AND MOISTURE

Light and moisture are two very important enviornmental factors besides others for the growth of microorganisms.  Apart from temperature, some microorganisms require light for reproductive cycle.

Moisture also play a povital role for microbial growth  it is required for the uptake and excretion of nutrients and waste materials.

SAMPLING TECHNIQUES

Before sampling is done, a plan has to be drawn to specifying which microorganims will be looked for, where they might be found, likely sources, how the organims will be located and quanitified, and when and where samples will be collected.

There are many kinds of volumetric air samplers, among which the Andersen sampler is frequently mentioned.  They draw in air from the room and discharge it with the bioaerosols onto a filter or sticky surfaces or agar plates.

Air samples can be used to get a count of “colony forming units” (cfu, which are bacteria and fungi that start growing on the culture media) per cubic meter of air.  If the colonies are easy to identify, the counts can be related to individual species.  The cfu of mixed species may also be used as a broad index of microbial growth.  When species are mixed, however, one or more species may be suppressed on the culture plate by competitors.

A well-known but unreliable method of taking a sample of bioaerosols in an area is to set out culture plates and let the spores and other bioaerosols settle on them.  This method is unreliable for the following reasons:

·      Some organisms do not send out many spores.

·      Some organisms have spores that are too light to settle on plates. 

·      Some species of organisms die soon after they leave the colony.

·      Settling plates will not collect respirable size particles (<10ì)

The mold count can be increased hundreds or thousands of times by “aggressive sampling”--that is, by stirring up the air with a fan, or pounding on the wall.  Higher counts are also had when there is traffic through a room, such as a schoolroom at the beginning or end of the day.  So a great many facts and observations have to be recorded, and samples have to be taken by several different methods, in oder to get reliable data.

Some environmental firms and labs have guidelines or background Bioaerosol numbers for building and homes.  These numbers are dependent on the laboratory’s qualifications, experience, capabilities and ability to query their database.

Samples of moldy materials (bulk samples) can be taken and pressed directly onto an agar plate, then cultured.  Results are counted as colony-forming units per gram of material.

Samples of surface growth can be taken with sticky tape or a sterile swab, then transferred to a plate.  This is a useful method for hard surfaces like the inside of an air duct.

For more information, please contact:

Vik Ahuja

Pure Air Control Services

(800) 422-7873, ext. 804