Microflora of soil, water and air


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Microflora of soil, water and air

  2. 2. Soil consists of mineral particles, air, water, soil organic matter, plantroots and living soil organisms. In terms of mass and volume, the livingorganisms constitute by far the smallest part of the soil.Typically, 50% of a soil volume will be mineral particles, 25% air filledpores and 25% water filled pores, although this will vary.The soil organic matter constitutes 0.5-5% of the solid fraction byweight, except in peat soils where it is much higher. Soil biota can be grouped according to size in micro-, meso- andmacrobiota.are less than 0.2mm and consist ofbacteria, actinomycetes, fungi, algae and protozoa.range from 0.2 to 10mm in size and consist ofnematodes, enchytraeids, collembola or springtails, mites, rotifersand small insects (arthropods).are organisms larger than 10mm and consist ofearthworms, mollusks and larger arthropods.
  3. 3. REASONS OF INVESTIGATION Epidemiological investigation Definition of transmission routes of diseases andresistance of their causative agents Pollution of soil waters, rivers, lakes etc. Definition of sanitary conditions of soil
  4. 4. Investigation of soil microbiota Investigation of microflora is difficultbecause of presence of many different species of microbes thatcan need totally different conditions for their isolation andidentification: temperature, respiration, nutrition etc. Because of that problem mostly are used indicating bacteriawhich show sanitary conditions of environment Indicating bacteria for soil: E.coli, Streptococcusfeacalis, Clostridium perfringens, bacteria of genus Proteus. In addition can be checked special pathogenic bacteria especiallyfor epidemiological reasons: Salmonellas, Shigellas, Clostridiumbotulinum and tetani etc.
  5. 5. Obtaining of samples Take samples from area close to possible pollution and on somedistance from it Take samples from 5 points by “envelope” type Samples must be close to 300 g to save moisture duringtransportation Transportation must be not more then 24 hours in temperature +4-5 C In laboratory soil primary must be cleared from stones, roots, leavesetc.
  6. 6. Obtaining of samples Should be performed in aseptic conditions with sterileinstruments Several probes of soil are multiplied in aseptic conditions andmixed with sterile water to make 10-1 dilution. (30g of soil with270 ml of water) Such suspension is used for preparation of other dilutionsaccording to method of investigation and approximate bacterialpopulation
  7. 7. Microbial number total amount of microorganisms in 1 g of soil “Clear” soils contain not more then 1-1,5millions of bacteria in 1g Results always must be correlated according tothe type of soil (different types of soil naturallycontains different amount of bacteria) Also undergo seasonal changes
  8. 8. Microbial number Often dilutions: 10-3 to 10-5 Not less then 2 dilutions must be used Mix solutions before seeding Each solution seed not less then 2 Petri dishes 1 ml of solution is taken, placed on a button of Petri dish Add 7-10 ml of boiled and cooled to 45 C MPA Mix MPA with suspension by soft rocking of Petri dish Mark dilution, data about investigation on Petri dish
  9. 9. Microbial number Incubation for 24 hours with temperature of 28-30 C For facility of cultivation better to take dilutions in which ondishes are formed from 50 to 150 colonies Protocols of calculation include such data:1. Area of investigation2. Amount of colonies obtained from several Petri dished accordingto dilutions3. Average amount of colonies4. Microbial number
  10. 10. Microbial number (example)Name of anareaAmount ofcolonies indilution 1:10 000Microbialnumber1. Yard ofkindergarten70= 8090800 000
  11. 11. Coli-titre and Coli-index Coli-titre – smallest amount ofsoil where present 1 E.coli Coli-index – amount of E.coli in 1g of soil For calculation of coli-index intocoli-titre: 1000 divide by coli-index
  12. 12. Titration method Mixture of 1:10 suspension with 50 ml of liquid nutrient media Mostly lactose broth with 1,5 ml of 2% water solution of TTC(2,3,5-triphenyl-2H-tetrazolium chloride) E.coli can reduce TTC to TPF (1,3,5-triphenylformazan) whichmake red-brown color. E.coli is resistant to TPF which break the growth of other bacteria Incubation for 24 hours with temperature of 37 C In presence of gas, changes of color of media to red-brown –seeding to Endo media
  13. 13. Titration method In presence on Endo media pink or red colonies of Gram”-” rodbacteria with negative oxidase activity it is performed theircalculation and results interpreted in a form of coli-titre For confirmation of results it is performed seeding of colonies onsemi-liquid media with glucose and incubation of it for 24 hourswith temperature of 37 C In presence in media acid and gas – results interpreted as positiveand confirmed
  14. 14. Titration method (other variant) Usage of Kessler medium (1% of the peptone, 5% of thebile, 0,25% of lactose and gentian-violet for inhibition Gram”+”bacteria) Incubation for 24-48 hours with temperature of 37 C In case of gas formation and opacity – seeding on Endo mediawith further investigation like in forehead method
  15. 15. Membrane filter method Can reduce time of analysis by 2 days because of exclusion of stageof cultivation on liquid medium For analysis of soil in small dilutions on membrane filter can beplaced plankton filter Calculation is performed on filters with 30-50 colonies After that performed calculations according to dilutions and numberof colonies
  16. 16. Direct superficial seeding method Used for investigation of “dirty” soils Soil suspensions with dilution 1:1 000 000 seed on Endo mediaand incubated for 24 hours with temperature of 37 C Calculation of pink or red colonies with metallic shining For more clear results these colonies undergo furtheridentification
  17. 17. Detection of Clostridium perfringens Soil dilutions (1:100 000) are placed by 1 ml into 2 rows of testtubes 1 row is heated 15 min with 80 C or 10 min with 90 C In all test tubes put 10 ml of boiled and cooled to 45 C Wilson-Blair medium (Bismuth Sulfite Agar) Typical Composition (g/liter):Meat extract 5.0; peptone from meat 10.0; D(+)glucose 5.0; di-sodium hydrogen phosphate 4.0; iron(III) sulfate 0.3; brilliantgreen 0.025; bismuth sulfite indicator 8.0; agar-agar 15.0 Spreading of suspension on medium and quick cooling in coldwater for removal of air
  18. 18. Detection of Clostridium perfringens Incubation even for 2 hours with 43 C In depth of agar appears black colonies which break mediumbecause of gas formation I smears must be detected Gram“+” rod bacteria Other variant: usage of SPN medium (sulphite-polimixyn-neomicyn medium) with incubation for 10-12 hours (temperature– 44-45 C)
  19. 19. Detection of Shigella and Salmonella1. Coagulation and centrifugation by Ficker From 30-50 g of soil prepare dilution 1:10 in sterile water For concentration of bacteria to 500 ml of suspension add 2 ml of10% solution of NaHCO3 and after that 1,7 ml of 10% solution ofFe2SO4 Mix suspension and leave it for 1 hour in temperature of 4 C Flakes of precipitation undergo centrifugation for 5 min andtitration with 25% tartaric potassium up to dilution ofsedimentation
  20. 20. Detection of Shigella and Salmonella Obtained solution undergo seeding on solid medium (Wilson-Blairmedium and Ploskirev’s medium) – 4 dishes Left solution filled with 50 ml of 10-20% yolk broth with furtherincubation (5-6 hours with 37 C) and inoculation to solid electivemedia After 8-20 hours – additional reseeding Further identification of bacteria performed according to classicalsteps of identification of Shigella and Salmonella
  21. 21. Detection of Clostridium tetani Obtained 20-30 g of soil multiplies by sterile instruments, 3-5 gof it mix with 10-15 ml of 0,9% solution of sodium chloride After 3-4 hours solution should be injected subcutaneously inright hind extremity of white mice (1 ml) Each probe is investigated in 2 mice For control are taken mice with forehead injection of antitoxicserum Death of experimental animals with symptoms of tetanus andsurvival of mice in control group confirms a presence of Cl. tetaniin soil
  22. 22. Detection of Clostridium botulinum 20-30 g of multiplied soil place in 80-100 ml of Kitt-Tarozzimedium 1 flask heat with 80 C during 30 min for killing of non-spore-forming bacteria Both flask incubate for 8-14 days (temperature – 37 C) Seed obtained material on sugar agar with further investigationaccording biological and antigenic properties of Cl. botulinum
  23. 23. OBTAINING OF SAMPLES Samples from open water take from depth of 10-15 cm fromsurface but not less then 10-15 cm from button Use Nansen bottle for that Tap water can be taken in sterile bottlewith volume 500 ml after 10 min od water flow andsterilization of pipe end with flame To chlorinated water it is necessary to add 2 ml of 1,5% solutionof sodium hyposulphate Transportation of samples must be in temperature +4-10 (6hours) or 2 hours without cooling
  24. 24. REASONS OF INVESTIGATION Sanitary control By epidemiological reason for detectionpathogenic intestinal bacteria(Salmonella, Shigella etc.), Enteroviruses … Detection of new fecal pollutions Choice of water source Checking of quality and level of clearness ofsewage water
  25. 25. Microbial number Investigation of total number of mesophilic aerobic andfacultative anaerobic bacteria in 1 ml of water that can in 24hours incubation in a temperature of 37 C cam form colonies onMPA which can be visible with eyes or 2-5 times zoom Depending on clearness of water prepare dilutions from 1:10 forclear water to 1:10 000 for very dirty sources For investigation of tap water use 1 ml without dilution Seed material on boiled and cooled to 45 C MPA or wort agar forfungi Incubate MPA for 24 hours (temperature 37 C), wort agar – 2-3days with temperature of 27 C
  26. 26. Microbial number Calculation performed with magnification on dishes with notmore then 300 colonies. If more – use other dilutions. Microbial number of tap water must be not more then 100 CFU(colony forming units) in 1 ml
  27. 27. Detection of E. coli: two phase fermentativetest This method differs from given in book!!! Correspond to Governmental Standard 18963-73 3x3 volumes of 10 ml, 1 ml and 0,1 ml – for 10 ml use flasks withlactose-peptonic medium, other – test tubes with 5 ml of medium For tap water – 3x3 volumes of 100 ml, 10 ml and 1 ml – for 100 mluse concentrated glucose-peptonic medium, for 10 ml and 1 ml –diluted one Cultivation for 24 hours, T – 38 C In case of absence of gas formation and precipitation – result isnegative
  28. 28. Two phase fermentative test In case of presence of gas formation and precipitation – materialseed on sectors of Endo medium for isolation of colonies If on Endo medium there is a growth of dark-red colonies withmetallic shining – perform oxidase test In presence of Gram”-” rod bacteria without oxidase – testrecognized as positive and interpreted in coli-index (number ofE.coli in 1 l of water) according to a table
  29. 29. Detection of new fecal pollution From 3 volumes of lactose-peptonic medium where afterincubation was found gas formation with a loop seed bacteria tolactose medium with boric acid Cultivation for 24 hours (T = 43 C) Presence of gas and opacity shows new fecal pollution Only opacity – result negative
  30. 30. Membrane filter method Filtration of water in volume 100, 10 and 1 ml for clear water and 0,1;0,01 ml for dirty water. Investigation stert from bigger dilutions For seeding volumes 1 ml and less primary mix it with 10 ml of sterilewater After filtration filters are taken with sterile forceps and placed on Endomedium (filtering surface on top): 1 dish – 3-4 membrane filters Incubation: 18-24 hours, T=37 C For calculation used filters with number of colonies from 10 to 50 For calculation of coli-index number of colonies multiplied by 1000 anddivided to a volume of investigated water Method detect more bacteria then two phase fermentative test!!!
  31. 31. Detection of Enterococci (Streptococcusfaecalis etc.) Index of Enterococci is defined according to cultivation in liquidalkaline polimixyn medium with 10-times dilutions depending onclearness of water (from 100 to 0,01 ml) 100 ml and 10 ml seed on double concentration of medium, rest –ordinary concentration Incubation: 24 hours, T=37 C Positive result – change of color, opacity For control from positive flasks and test tubes bacteria seed ondishes with milk-inhibitor medium. Streptococcus faecalis formthere black colonies with metallic shining
  32. 32. Detection of pathogenic bacteria1. SalmonellaPrimary seeding on accumulation media (magnesium, seleniticmedium). Further investigation goes according classical forSalmonellas method2. ShigellaPerformed on tap water in cases of accidents with sewage system.For accumulation media is used wort media (400 ml of water mixwith 100 ml of wort medium)After incubation for 24 hours (T=37 C) material seed on Ploskirev’s orLevin’s media with further classical identification
  33. 33. REASONS OF INVESTIGATION Definition of bacterial pollution of air withmicrobes from nosopharynx of humans Direct investigation of presence of pathogenic andconditionally pathogenic bacteria as causativeagents of nosocomial infections On factories: investigation of presence in airmicrobes that are used for industrial reasons
  34. 34. Koch’s Method(sedimentation method) Set open Petri dishes with MPA in a room for 10 min (for cocci – 40min, special media) Incubation: 24 hours (T=37 C) and 24 hours (room temperature) Calculate number of colonies, measure diameter of dish For calculation of microbial number (amount of bacteria in 1 m3 of air):number of colonies multiply by coefficientDiameter of dish, cm Area of dish, sq cm Coefficient for 10 minexposure8 50 1009 63 8010 78 60
  35. 35. Krotovs method (aspiration method) More sensitive because not dependent onairflow in room With a help of centrifugal fan air is absorbedthrough a fissure and spread of rotating Petridish with a medium. Speed – 20-25 m/min; timeof exposure – 2 min Incubation: 24 hours (T=37 C) and 24 hours(room temperature) Calculation of microbial number: amount ofcolonies multiply by 1000 and divide by volumeof absorbed air
  36. 36. Krotovs method (aspiration method)1. Detection of Staphylococci250 dm3 of air absorb by Krotov’s apparatus on 203 dishes with milk-yolk-salt agar and blood agarIncubation: 37 C , 48 hours.2. Detection of Streptococci200-250 dm3 of air absorb by Krotov’s apparatus on 203 dishes withGarro medium and blood agarIncubation: 37 C , 18-24 hours, after that 48 hours in roomtemperature