MEDICAL MICROBIOLOGY I LECTURE 2 Culture of Bacteria and Bacterial Growth
Culture Media • Microbiology depends on the ability to GROW and MAINTAIN microorganisms in the laboratory - use of suitable culture media • A culture medium is a solid or liquid preparation used to grow, transport, and store microorganisms. • They must contain water and sources of nitrogen, carbon, mineral salts and essential vitamins. • Some bacteria may require additional specific substances which may be added to the medium.
Culture Media • Two categories of culture medium: 1. Chemically defined media • •
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Concentration of each ingredient is known Made of highly purified inorganic salts and simple organic compounds such as glucose or purified amino acids Little or no difference in composition between batches Expensive and not for routine use Used to determine specific growth requirements of bacteria
Culture Media 2. Complex Media • • • • •
Media prepared using natural products such as meat extract or vegetable infusions. Natural products contain essential bacterial nutrients Exact concentration of nutrients are unknown Used routinely Easy to prepare, relatively cheap and able to support the growth of many bacteria
Types of Media 1. Liquid media or broth – Bacteria can move freely in them – Growth in liquid medium is shown by turbidity – Some organism show surface growth – Used for biochemical testing, blood culture, testing for motility and as enrichment broth – Disadvantage: purity of growth cannot be guaranteed
Types of Media 2. Solid Media – Microorganism grown on solid media will grow and multiply at the site of inoculation and form visible colonies – Liquid agar is made solid by adding a solidifying agent which does not alter the nutritional content of the medium - agar – Agar is an inert carbohydrate extract obtained from a type of seaweed found in Japan, New Zealand, and California.
Types of Media • Properties of agar: 1. 2. 3. 4. 5. 6. 7. 8.
Melts at 98°C, Sets at around 40°C, Easily soluble Remains clear Concentration of 1% to be gel. It is solid at 37°C Once solidified, it can be remelted Bacteriologically inert (will not be degraded by most bacteria)
Types of Media 3. Basal Media – Simple media that will support the growth of most microorganisms that do not need special nutritional requirements – Contain basic nutrients: peptone, mineral salts and water – Normally called nutrient broth – 2 types: infusion broth and digest broth
Types of Media 4. Enriched media – Culture media that are enriched with whole or lysed blood, serum, special extracts or nutrients – Support growth of bacteria that cannot grow on basal media – Nutrient broth + agar = nutrient agar – Nutrient agar + blood = blood agar – Blood agar + heat = chocolate agar
Types of Media 5. Selective media – Solid media has substances that prevent, slow down or inhibit the growth of microorganisms other than those for which the media are devised e.g. • •
tellurite medium for diptheria organism Deoxycholate citrate agar (DCA) for Salmonella and Shigella groups
Types of Media 6. Enrichment media – Liquid media, similar in function to selective media – Difference: selective media is broth – e.g. selenite F broth for the isolation of Salmonella group
Types of Media 7. Differential media – Contain substances or indicators that will differentiate one organism from another – e.g. MacConkey agar - differentiate lactose fermenting bacteria from non-lactose fermenting bacteria; blood agar - differentiate haemolytic bacteria from non-haemolytic ones.
Types of Media 8. Transport media – Usually semi-solid – Transportation of clinical specimens containing delicate microorganisms, if there is to be a delay in their delivery to the laboratory or in processing – Contain substances that can prevent the overgrowth of commensals and prevent bacteria from dying as a result of pH change or enzyme action, e.g. • •
Amies transport medium for Neisseria gonorrhoeae Stuart’s transport medium for delicate organisms including anaerobes
Isolation of Pure Cultures • In their natural habitats, microorganisms usually grow in complex, mixed populations with many other species. • Pure culture - a population of cells arising from a single cell to characterise an individual species • Approaches: 1. Spread plate and streak plate 2. Pour plate 3. Microbial growth on agar surfaces
Mixed Bacterial Growth
Isolation of Pure Cultures 1. Spread plate and streak plate – Mixture of cells is spread out on agar surface at a relatively low density. – Every cell grows into a completely separate colony, a macroscopically visible growth of clusters of microorganism on a solid medium.
Isolation of Pure Cultures I. Spread plate –
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Small volume of dilute microbial mixture containing around 30 - 300 cells is transferred to the center of agar plate and spread evenly over the surface with a sterile bent-glass rod. Dispersed cells develop into isolated colonies The number of colonies should equal the number of viable organism in the sample. Spread plates can be used for colony count.
Isolation of Pure Cultures II. Streak plate –
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The microbial mixture is transferred to the edge of an agar plate with an inoculating loop or swab and then streaked out over the surface in one of several pattern. After the first quarter is streaked, the inoculating loop is sterilised and an inoculum for the second quarter is obtained from the first quarter. Similar process is repeated for the subsequent quarters. This is essentially a dilution process to separate individual colonies.
Streak Plate
Isolation of Pure Cultures 2. Pour plate – After the agar solidifies, each cell is fixed in place and forms an individual colony – Plates containing 30 -300 colonies are counted – The colonies equal the number of viable microorganism in the sample that are capable of growing in the medium used – Colonies growing on the surface also can be used to inoculate fresh medium and prepare pure cultures
Pour Plate
Isolation of Pure Cultures 3. Bacterial growth on agar surfaces – Colony development on agar surfaces aids microbiologists in identifying microorganisms because individual species often form colonies of characteristic size and appearance – In nature, microorganisms often grow on surfaces in biofilms - slime-encased aggregations of microbes – Sometimes they form discrete colonies
Isolation of Pure Cultures • Generally the most rapid cell growth occurs at the colony edge • Growth is much slower in the center, and cell autolysis takes place in the older central portions of some colonies. • These differences in growth are due to gradients of oxygen, nutrients, and toxic products within the colony.
Isolation of Pure Cultures • At the colony edge, oxygen and nutrients are plentiful • The colony center is much thicker than the edge • Oxygen and nutrients do not diffuse readily into the center, toxic metabolic products cannot be quickly eliminated, and growth in the colony center is slowed or stopped
Isolation of Pure Cultures • Cells on the periphery can be growing at maximum rate while cells in the center are dying • The bacteria growing on solid surfaces vary with nutrient diffusion and availability, the hardness of the agar surface, bacterial chemotaxis, and the presence of liquid on the surface
Isolation of Pure Culture • Tube culture methods – Used for specific identification tests – Only small volumes of media are required for the tests – Not used for colonial morphology study – e.g. are: 1. Slope cultures 2. Deep cultures 3. Stab cultures
Isolation of Pure Culture • Slope (slant) cultures are tubes containing small quantity of medium that has been allowed to set in sloped position. – Aka ‘slopes’ or ‘slants’ – Used for maintenance of isolated bacteria or for performing biochemical tests – e.g. Loeffler's serum agar, Dorset egg medium and Lowestein-Jensen medium
Isolation of Pure Culture • Deep culture media are prepared in tubes of about 150 x 20 mm to a depth of about 60-70 mm. – Aka ‘shake’ – Cultivation of anaerobic bacteria and can be used for viable count
• Stab cultures refer to the method of inoculation, where the inoculating needle is stabbed through the center of the medium. – e.g. Kohn’s II medium, motility medium, indole urea medium
Isolation of Pure Culture • Inoculation into broth – Carried out using inoculating loop, inoculating needle or Pasteur pipette – Depends on whether the inoculum is colonial growth, liquid culture or specimen – Pick colonies with sterile inoculating loop, hold the tube at an angle, and rub the loop against the inner side of the container below the level of the broth
Sub-culturing • Sub-culturing means to transfer microorganism from one medium to another. • Basically, it is starting new bacterial cultures from the old cultures. • Usually performed to maintain continuous growth of microbes
Measurement of Microbial Growth • To determine growth rates and generation times • Either population number or mass may be followed because growth leads to increases in both • No single technique is always best, the most appropriate approach will depend on the experimental situation
Measurement of Microbial Growth • Classified into 2 methods: 1. Cell number i. ii. iii. iv.
Direct counting Coulter counter and flow cytometer Membrane filter technique Plating methods (visible counting methods, spread plate and pour plate methods)
2. Cell mass i. Microbial dry weight ii. Spectrophotometry
Measurement of Microbial Growth 1. Direct counting – Using counting chamber e.g. Petroff-Hausser counting chamber, haemocytometers – Prokaryotes are more easily counted when stained, or if phase contrast or flourescence microscope is used – Advantage: easy, inexpensive, and relatively quick; it gives information about the size and morphology of organisms – Disadvantage: the microbial population must be fairly large for accuracy because only a small volume is sampled
Petroff-Hausser Counter
Measurement of Microbial Growth 2. Coulter chamber and flow cytometer – Large microorganisms like protists and yeasts can be directly counted with electronic counters, Coulter chamber and flow cytometer – The microbial suspension is forced through a small hole or orifice in the Coulter counter – An electrical current flows through the hole, and electrodes placed on both sides of the orifices measure its electrical resistance
Coulter Counter
Measurement of Microbial Growth • Every time a microbial cell passes through the orifice, electrical resistance increase (or conductivity drops) and the cell is counted • Advantage: accurate with large cells and extensively used • Disadvantage: interference by small particles, the formation of filaments and other problems
Measurement of Microbial Growth 3. Membrane filter techniques – The number of bacteria in aquatic samples is frequently determined from direct counts after the bacteria have been trapped on special membrane filters – Sample is first filtered through a black polycarbonate membrane filter. – Then, the bacteria are stained with a fluorescent dye and observed microscopically
Measurement of Microbial Growth – Stained cells are easily observed against the black background of the membrane filter and can be counted when viewed with an epifluorescence microscope – Traditional counting methods do not distinguish dead cells from live cells – New methods (e.g. commercial kits) make this possible – First traps bacteria in aquatic samples on a membrane filter
Measurement of Microbial Growth – The filter is then placed on an agar medium or on a pad soaked with liquid media and incubated until each cell forms a separated colony – A colony count gives the number of microorganisms in the filtered sample, and special media can be used for specific microorganisms – Specially useful technique in analysing water purity
Measurement of Microbial Growth 4. Plating methods – To determine the number of viable microbes – Viable counting methods - count only those cells that are alive and able to reproduce – Pour plate and spread plate methods - diluted samples of bacteria or other microorganisms is dispersed over a solid agar surface – Simple, sensitive, and widely used for viable counts
Measurement of Microbial Growth – Low counts will result if clumps of cells are not broken up and microorganisms not well dispersed – Results expressed in colony forming units (CFU) 30 - 300 colonies for most accurate counting
Measurement of Microbial Growth 5. Microbial dry weight – Cells growing in liquid medium are collected by centrifugation, washed, dried in an oven and weighed – Especially useful technique for measuring the growth of filamentous fungi – Time-consuming and not very sensitive – Bacteria weigh so little, it may be necessary to centrifuge several hundred millimeters of culture to collect a sufficient quantity
Measurement of Microbial Growth 6. Spectrophotometry – More rapid and sensitive – Depend on the fact that microbial cells scatter light that strikes them – Microbial cells in a population are of roughly constant size – The amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number
Measurement of Microbial Growth – When the concentration of bacteria reaches about 107/mL, the medium appears slightly cloudy or turbid – The population growth can be easily measured as long as the population is high enough to give detectable turbidity
Measurement of Microbial Growth • If the amount of a substance in each cell is constant, the total quantity of that cell constituent is directly related to the total microbial cell mass • Example, a sample of washed cells collected from a known volume of medium can be analysed for total protein or nitrogen • An increase in the microbial population will be reflected in higher total protein levels. • ATP can be used to estimate the amount of living microbial mass
