Lesson 46
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 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:
- 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.
- 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.
- 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-titreis 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.
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 ofwater. 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 Bacteriophagesof 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 m 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
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.
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 = r2 =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 standardsof 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.
Lesson 48
Theme: Virology.
Morphology and structure of the viruses.
Methods of their cultivation.
I. STUDENTS’ INDEPENDENT STUDY PROGRAM
1. Classification, structure and chemical composition of viruses:
a – basis principles of classification of viruses; modern classification of viruses.
b – structure of a virion, their dimensions; simple (naked) and complex (enveloped) viruses;
c – types of virus symmetry;
d –chemical composition of viruses
2. Main methods of cultivation of viruses:
a – inoculation of a laboratory animal;
b – inoculation of a embryonated eggs;
c – inoculation into the cell cultures, their types and classification.
3. Types of interaction of viruses and sensitive cells.
4. Replicative cycle of a virus in the host cell:
5. Prions and viroids as causative agents of different diseases. Their biological properties.
Viruses are the smallest infectious agents (20-300 nm in diameter), containing of the one kind of nucleic acid (RNA or DNA) as their genome, usually as single molecule. The nucleic acid is enclosed in a protein shell, and the entire infectious unit is termed a virion. Viruses replicate only into the living cells.
Some Useful Definitions in Virology
Capsid: The symmetric protein coat (shell) that encloses the nucleic acid genome.
Nucleocapsid: The capsid together with the enclosed nucleic acid.
Capsomeres: Capsomeres represent clusters of polypeptides, which form the capsid.
Virion: The complete infective virus particle, which in some instances (adenoviruses, papovaviruses, picornaviruses) may be identical with the nucleocapsid. In more complex virions (herpesviruses, myxoviruses) it includes the nucleocapsid plus a surrounding envelope.
Defective virus: A virus particle that is functionally deficient in some aspect of replication. Defective virus may interfere with the replication of normal virus.
Evolutionary origin of viruses. The origin of viruses is not known. Three hypotheses have been proposed:
(1) Viruses became parasites of primitive cells, and they evolved together. Many viruses today cause no host cell damage and remain latent in the host.