Microbes Ecology, Microflora & Sanitary-indicative bacteria of soil, water & Air

1. MICROBES ECOLOGY. MICROFLORA AND SANITARY-INDICATIVE
BACTERIA OF SOIL, WATER, AIR. THE METHODS OF STUDYING
I. THEORETICAL QUESTIONS
1. The main representatives of microflora of soil. The diseases transmitted by soil.
Sanitary - exponential species. Methods for studying of soil microflora.
2. The main representatives of microflora of the water. The diseases transmitted by water.
Sanitary - exponential species.
3. The methods of studying of microbial number and coli-index of the water. The
specifications of state standard.
4. The main representatives and sanitary - exponential species microflora of the air. The
diseases transmitted by air.
5. The methods of studying of air microflora (methods of sedimentation and aspiration).
Microbes are distributed everywhere in the environment surrounding us. They are found in
the soil, water, air, in plants, animals, food products, various utensils, in the human body, and on
the surface of the human body.
The relationship of micro-organisms with the environment has been named ecology (Gr.
oikos home, native land, logos idea, science).
Soil Microflora
Soil fertility depends not only on the presence of inorganic and organic substances, but
also on the presence of various species of micro-organisms which influence the qualitative
composition of the soil. Due to nutrients and moisture in the soil the number of microbes in 1 g
of soil reaches a colossal number — from 200 million bacteria in clayey soil to 5 thousand
million in black soil. One gram of the ploughed layer of soil contains 1-10 thousand million
bacteria.
Soil microflora consists algae (nitrifying nitrogen-fixing, denitrifying), cellulose-splitting
and sulfur bacteria, pigmented microbes fungi, protozoa, etc.
The greatest amount of microbes (1 000000 per cu cm) is found in the top layer of soil at a
depth of 5-15 cm. In deeper layers (1.5-5 m) individual microbes are found. However, they have
been discovered at a depth of 17.5 m in coal, oil, and artesian water.
The number of microorganisms in the soil depends on the extent of contamination with
faeces and urine, and also on the nature of treating and fertilizing the soil.
Saprophytic spores (B. cereus. B, meguterium, etc.) survive for long periods in the soil.
Pathogenic bacteria which do not produce spores due to lack of essential nutrients, and also as
a result of the lethal activity of light, drying, antagonistic microbes, and phages do not live
long in the soil (from a few days to a few months)
Usually the soil is an unfavourable habitat for most pathogenic species of bacteria,
rickettsiae, viruses, fungi, and protozoa. The survival period of some pathogenic bacteria is
shown in Table 2. However, the soil as a factor of transmitting a number of causative agents of
infectious diseases is quite a complex substrate. Thus, for example, anthrax bacilli after falling
on the soil produce spores which can remain viable for many years.
As is known, the spores of clostridia causing tetanus, anaerobic infections, and botulism,
and of many soil microbes survive for long periods in the soil. The cysts of intestinal protozoa
(amoeba, balantidium, etc.) spend a certain stage in the soil. The soil plays an important role in
transmitting worm invasions (ascarids, hook-worms, nematode worms, etc.). Some fungi live in
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2. the soil. Entering the body they cause fusariotoxicosis, ergotism, aspergillosis, penicilliosis
mucormycosis, etc.
Taking into consideration the definite epidemiological role played by the soil in spreading
some infectious diseases of animals and man, sanitary-epidemiological practice involves
measures directed at protecting the soil from pollution and infection with pathogenic species of
microorganisms.
A valuable index of the sanitary condition of the soil is the discovery of the colibacillus
and related bacteria, also enterococci, and Clostridium perfringens. The presence of the latter
indicates an earlier faecal contamination.
Microbiological investigation of soil. For this purpose it is necessary to select most
typical area not more then 25 m2
. The samples are taken from different places of the are field
along the diagonal, the angles and the center 10 — 20 cms deep. The weight of each sample
must be 100 – 200 g. The total weight of the soil 0,5 – 1 kg.
After careful mixing take an average sample of weight 100 – 200 g. Put the samples of soil
in the sterile banks, mark and deliver to the laboratory. The soil specimens for plating are
grinded in sterile mortar, make serial dilutions in an isotonic solution of sodium chloride 1: 10,
1:100, 1: 1000 etc. Plate 0,1 – 1 ml of specimens into special media for aerobic and anaerobic
microbes. After incubation at optimal temperature count the colonies on the plates.
Microflora of the Water
Pseudomonas fluorescens, Micrococcus roseus, etc., are among the specific aquatic
aerobic microorganisms. Anaerobic bacteria are very rarely found in water.
The microflora of rivers depends on the degree of pollution and the quality of purification
of sewage waters flowing into river beds. Micro-organisms are widespread in the waters of the
seas and oceans. They have been found at different depths (3700-9000 m).
The degree of contamination of the water with organisms is expressed as saprobity which
designates the total of all living matter in water containing accumulations of animal and plant
remains. Water is subdivided into three zones:
1. Polysaprobic zone is strongly polluted water, poor in oxygen and rich in organic
compounds. The number of bacteria in 1 ml reaches 1 000000 and more.
Colibacilli and anaerobic bacteria predominate which bring about the processes of
putrefaction and fermentation.
2. In the mesosaprobic zone (zone of moderate pollution) the mineralization of
organic substances with intense oxidation and marked nitrification takes place. The
number of bacteria in 1 ml of water amounts to hundreds of thousands, and there is
a marked decrease in the number of colibacilli.
3. The oligosaprobic zone is characteristic of pure water. The number of microbes is
low, and in 1 ml there are a few tens or hundreds; this zone is devoid of the
colibacillus.
Tap water is considered clean if it contains a total amount of 100 microbes per ml,
doubtful if there are 100-150 microbes, and polluted if 500 and more are present. In well water
and in open reservoirs the amount of microbes in 1 ml should not exceed 1000. Besides, the
quality of the water is determined by the presence of E. coli and its variants.
The degree of faecal pollution of water is estimated by the colititre or coli-index. The coli-
titre is the smallest amount of water in millilitres in which one E. coli is found. The coli-index is
the number of individuals of E.coli found in 1 litre of water. Tap water is considered good if the
coli-titre is within the limits of 300-500. Water is considered to be good quality if the coli-index
is 2-3.
Due to the fact that Str. faecalis (enterococci) are constant inhabitants only of the intestine
in man and warm-blooded animals, and are highly resistant to temperature variations and other
environmental factors, they are taken into account with the coli-titre and coli-index for the
determination of the degree of faecal pollution of water, sewage waters, soil, and other objects.
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3. Water is an important factor for the transmission of a number of infectious diseases
(enteric fever, paratyphoids, cholera, dysentery, leptospiroses, etc.).
Due to the enormous sanitary-epidemiological role of water in relation to the intestinal
group of diseases, it became necessary to work out rapid indicator methods for revealing
colibacillus and pathogenic bacteria in water.
These include the methods of luminescent microscopy for the investigation of water for the
presence of pathogenic microbes and the determination of the increase of the titre of the phage.
Upon the addition of specific phages to liquids containing a homologous microbe in 6-10 hours a
considerable increase in the amount of phage particles can be observed.
Microbiological investigation of water. The sanitary - bacteriological investigation of
water includes determination of total number of microbes in 1 ml of water, determination of a
coli-index or coli-titer, and detection of pathogenic microbes, and Bacteriophages of Е. соli.
Quantitative Analysis. The employed method is the plate count. A measured volume of
water is serially diluted (see below), following which 1 mL from each dilution tube is seeded in
nutrient agar and the then colonies counted.
A typical example of serial dilution should be made by following way. One millilitre of the
water sample is aseptically transferred by pipette to 9 mL of sterile water. (For obvious reasons,
this is also known as the "10–1
" dilution). The process is repeated serially until a dilution is
reached that contains between 30 and 300 colony-forming cells per millilitre. Several samples
(1-mL of appropriative dilution) are plated in a nutrient medium. Since the original sample may
have contained up to 1 million (106
) viable bacteria, it is necessary to dilute all the way to 10–5
,
plate 1-mL samples from each dilution tube, and then count the colonies only on plates
containing 30-300 colonies.
The drinking water should not have more than 100 microbes in 1 ml. The microbial
number in water of wells and open reservoirs can be up 1000.
Qualitative Analysis.
2. Membrane filtration method. A large measured volume of water is filtered through a
sterilized membrane. Filter retains bacteria on its surface (fig.1). The membrane is then
transferred to the surface of an agar plate containing a selective differential medium for coliform
bacteria (fig. 2). Upon incubation, coliform bacteria give rise to typical colonies on the surface of
the membrane.
Water samples (100 ml) are passed through bacteriological filters (0,2 to 0,45 µm pore size)
to trap bacteria The filters with trapped bacteria are placed on a medium containing lactose as
a carbon source, an inhibitor to suppress growth of noncoliforms and indicator substances to
facilitate differentiation of coliforms. Coliform bacteria form distinct colonies on Endo
medium
During determination of a coli-index and coli-titre of water it is necessary to take into
consideration the ability of Е. coli of the man and animal to grow at 43 °C
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4. Microflora of the Air
The composition of the microbes of the air is quite variable. Then more dust, smoke, and
soot in the air, the greater the number of microbes. Each particle of dust or smoke is able to
adsorb on its surface numerous microbes.
The number of microbes in the air varies from a few specimens to many tens of thousands
per 1 cu m. Pathogenic species of microbes (pyogenic cocci, tubercle bacilli, anthrax bacilli,
bacteria of tularaemia, rickettsiae of Q-fever, etc.) may be found in the surroundings of sick
animals and humans, infected arthropods and insects, and in dust.
At present Streptococcus viridans serves as sanitary indices for the air of closed buildings,
and haemolytic streptococci and pathogenic staphylococci are a direct epidemiological hazard.
Depending on the time of the year, the composition and the amount of microflora change. If the
total amount of microbes in winter is accepted as 1, then in spring it will be 1.7, in summer— 2
and in autumn — 1.2.
The total amount of microbes in an operating room before operation should not exceed 500
per 1 cu m of air, and after the operation not more than 1000. There should be no pathogenic
staphylococci and streptococci in 250 litres of air. In operating rooms of maternity hospitals
before work the number of saprophyte microflora colonies isolated from the air by
precipitating microbes on meat-peptone agar within 30 minutes should not exceed 20.
The number of microbes in factories and homes is associated closely with the sanitary
hygienic conditions of the building. At poor ventilation and natural lighting and if the premises
are not properly cleaned, the number of microbes increases.
The causative agents of influenza, measles, scarlet fever, diphtheria, whooping cough,
meningococcal infections, tonsillitis, acute catarrhs of the respiratory tract, tuberculosis,
smallpox, pneumatic plague, and other diseases can be transmitted through the air together with
droplets of mucus and sputum during sneezing, coughing, and talking.
The air is an unfavourable medium for microbes. The absence of nutrient substances, the
presence of moisture, optimal temperature, the lethal activity of sunlight, and desiccation do not
create conditions for keeping microbes viable and most of them perish. However, the relatively
short period during which the microbes are in air is quite enough to bring about the transmission
of pathogenic bacteria and viruses from sick to healthy persons, and to cause extensive
epidemics of diseases such as influenza.
The laboratory investigation of air is carried out to determine the qualitative and
quantitative composition of its microflora. This is achieved by using simple and complex
methods. For a more accurate investigation of microbial contents of the air special apparatus are
used.
Microbiological investigation of the air. The sanitary - hygienic investigation of the
microflora of the air includes determination both the total number of microbes in 1 m3
of the air
and revealing of pathogenic staphylococci and streptococci. For taking the samples
sedimentation and aspiration methods are used.
Plate method (sedimentation method). The Petri’s dishes with meat-peptone agar or
another special nutrient media for staphylococci and streptococci, for example blood agar, yolk-
salt agar are used. They are opened and are stayed in investigated room. Term of exposition
depends on prospective quantity of microbes in the air. With a plenty of microorganisms a plate
is opened for 5 – 10 minutes, with a little – for 20 — 40 minutes.
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5. Then the dishes put into thermostat at 37 °C for 24 hrs. After incubation all colonies are
accounted (for determination of total number of microorganisms).
According to Omeliansky’s data in 5 minutes on a surface of 100 cm2
so many microbes
sedimentate, as they present in 10 L of air. For example, on the dish surface with MPA after 5
minute exposure 32 colonies have grown. It is necessary to calculate amount of microbes which
are present in 1 m3
of the air, applying the Omeliansky’s formula. The plate has 63 cm2
(S = πr2
=3,14 • 4.52
= 63 см2
). Thus, it is possible to determine, what quantity of microbes (х) would
grow at the given exposure on a surface of medium in 100 см2
,
x = (32 • 100) : 63 = 51
This quantity of microbes contains in 10 L of the air, and in 1 m3
(1000 л) there will be –
(51 • 1000) : 10 = 5100.
For determination of microbial dissemination degree quantity of the colonies on the dish
surface which have been counted should be multiplied with one of multiplier.
Aspiration method. Krotov’s apparatus is used for this purpose. It give us the possibility
to let pass 50 –100 L of air with a speed of 25 L per minute through clinoid chink in the special
glass above the open dish with MPA. The rotation of Petry’s dish (1 rotation/sec) provides
uniform dispersion of microorganisms on all surface of a medium. Then dish is incubated in a
thermostat at 37 °C for 18-24 hrs.
For example, 250 colonies are revealed on the surface of dish after 2-minutes exposure with a 25
l/min speed. Thus the number of microbes (x) in 1 l of the air is: x = (250 • 1000) : 50 = 5000.
There are temporary standards of a sanitary - hygienic state of the air: in operating
room the total number of microbes prior to the beginning of the operation must be no more than
500 in 1 m3
, after the operation – 1000.
In preoperative and dressing rooms limiting number of microbes prior before the
beginning of work – 750 microbes in 1 m3
, after work – 1500. In birth wards the total number of
microbes is about 2000 in 1 m3
of the air, and staphylococci and streptococci are not higher then
24 in 1 m3
, and in newborn rooms – about 44 in 1 m3
.
ІI. Students Practical activities
1. Determine the total number of microorganisms in water.
Account the colonies on the surface and within MPA.
Recording dilution determine the total number of microbs in 1 ml of seeding
water.
Make a conclusion about saprobity of water.
2. Determine the total number of microorganisms in 1 m3
of the air (exposure time is 5
minutes, speed is 25 l/min). Compare with normative data and make a conclusion.
3. Determine the number of bacteria in 1 m3
of the air by sedimentation method.
Account the colonies that have growth on the surface of MPA.
Account the total number of microorganisms in air using Omeliansky’s formula
(exposure is 5 min).
Compare with normative data and make a conclusion.
4. Determine a coli-titer and coli-index of water using method of membrane filters.
Account coliform colonies on the filter.
Determine a coli-titer and coli-index of water (the volume of water passed
through the filter is 500 ml (sample 1); is 100 ml (sample 2); is 1000 ml (sample 3))
Compare with normative data for tap water and make a conclusion about quality
of water.
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