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Techniques in basic microbiology Thanut Amatayakul Faculty of Agricultural Product Innovation and Technology Srinakharinwirot University Outline • • • • • Aseptic techniques Sterilization Microscopic study Microbial Quantification Media types and preparation 21/12/54 AIT 191 Microbiology 2 ASEPTIC CONCEPT AND TECHNIQUE 21/12/54 AIT 191 Microbiology 3 Aseptic • Processes or activities carried out under sterile condition • Prevent contamination – From environment – To environment • How??? 21/12/54 AIT 191 Microbiology 4 Working area and equipment surface disinfection • • • • Treating surface of working area with disinfectant Destroying vegetative cells and virus Endospore is not destroyed Disinfectant – – – – – – 21/12/54 70% ethyl alcohol | isopropanol NaOCl 200 ppm Fomaldehyde | Glutaraldehyde Iodine Phenol Etc. AIT 191 Microbiology 5 Mechanism-Example • Ethanol and isopropyl alcohol – Solubilize lipid in cell wall – Denature and precipitate protein inside microbial cell • NaOCl (sodium hypochlorite) 6000 ppm – oxidises sulphydryl (−SH) and – disulphide (S−S) bonds in proteins – Corrosive to stainless steel 21/12/54 AIT 191 Microbiology 6 Mechanism Name Effective dose Mechanism Glutaraldehyde Phenol Iodine Hydrogen peroxide Ethylene dioxide Ozone UV-C 21/12/54 AIT 191 Microbiology 7 21/12/54 AIT 191 Microbiology 8 21/12/54 AIT 191 Microbiology 9 Effectiveness • • • • • • Concentration Treating time Temperature Sensitivity of microbial Microbial number Nutrient availability 21/12/54 AIT 191 Microbiology 10 Aseptic-Microbial transfer 21/12/54 AIT 191 Microbiology 11 Aseptic 21/12/54 AIT 191 Microbiology 12 STERILIZATION 21/12/54 AIT 191 Microbiology 13 Definition of Sterilization • Sterilization: processes that are used to eliminate (remove and kill) microorganism as well as endospore on the surface as well as in the fluid • Means – Heat (dry & wet) – Chemical (liquid/gas) – Irradiation – Filtration – Pressure 21/12/54 AIT 191 Microbiology 14 21/12/54 AIT 191 Microbiology 15 Filtration-sterilization • Membrane with pore size 0.22 mm 21/12/54 AIT 191 Microbiology 16 Air filter • Air can be cleaned by passing through HEPA filter (High Efficiency Particle Air) • It is mostly used in biological safety cabinet • Removing particle < 0.3 mm at 99.97% 21/12/54 AIT 191 Microbiology 17 21/12/54 AIT 191 Microbiology 18 Moist heat • • • • • • • • Steam under pressure (high pressure) 121oC for 15 min/135oC for 40 min Equipment : retort/autoclave Water boil at 100oC at atmospheric pressure to achieve > 100oC need to increase pressure above water Increase pressure inside autoclave chamber to 103 kPa or 15 lb/in2 increase temperature to 121oC To kill spongiform encephalopathies (prions) need higher temperature or longer time at 121oC More effective than dry heat High heat coagulation and denaturation of protein of microbial 21/12/54 AIT 191 Microbiology 19 Be careful !!! It like a boom ที่มา: http://www.surge.com.tw/online% 20catalog/online/10.htm ที่มา : Hogg (2005) 21/12/54 AIT 191 Microbiology 20 ที่มา : Hogg (2005) 21/12/54 AIT 191 Microbiology 21 Precaution!!! • It is hot. • It can be explosive. • Confirm that pressure come back to atmospheric pressure before open the vessel. • Bottle or flask must not be over filled | just 2/3 at most 21/12/54 AIT 191 Microbiology 22 Dry heat • Heat article (metallic) in hot air oven at 160oC for at least 1 hour • It works by burning the cell components • Material should be dried before putting in the chamber • The chamber should not be overcrowded. (air flow) • The oven should not be open until the end of sterilizing cycle to prevent breakage of glassware 21/12/54 AIT 191 Microbiology 23 Sterilization by UV (non-ionizing radiation) • UV ray: 200-280 nm • 260 nm is the most effective for sterilization high-pressure mercury vapor lamp • Induce formation of thymine-thymine dimer inhibiting DNA replication | it do not kill spore 21/12/54 AIT 191 Microbiology 24 ที่มา: http://www.medimagery.com/pathology/bacteria.html 21/12/54 AIT 191 Microbiology 25 UV ray • • • • • • Do not kill spore Low penetration Limit life of UV bulb Clean surface without dust Harmful to skin and eye However, It doesn't penetrate glass, paper or plastic. 21/12/54 AIT 191 Microbiology 26 Other vocabulary • Sanitization: Processes of removing microorganism on the surface of utensils by either chemical or mechanical means to the level of safety • Asepsis : techniques to achieve microbial free environment • Antiseptic : a chemical mean to eliminate pathogenic microorganisms from skin • Bacteriostasis : a condition that bacteria is not multiply, but it is not death • Bacteriocidal : a condition that kill or inactivate bacteria 21/12/54 AIT 191 Microbiology 27 MICROSCOPIC STUDY 21/12/54 AIT 191 Microbiology 28 Light microscope • • • • • Bright field Dark field Polarizing Phase contrast Fluorescence ที่มา Benson (2001) 21/12/54 AIT 191 Microbiology 29 ที่มา http://www.visualphotos.com/image/1x3744243/paramecium_bright_field_light_micrograph_of 21/12/54 AIT 191 Microbiology 30 Compound light microscope • Transmission-thin section of sample • Objective lens: provides a magnified and inverted image of the specimen 21/12/54 • Eyepiece lens: adds further magnification • Condenser: focuses light onto the specimen to give optimum illumination (under stage) • Magnification: 4x to 100x • Diaphram: regulate the amount of light passing through the slide AIT 191 Microbiology 31 Resolution • It is the resolving power (d) of lens • Or the ability to resolve between Two points a short distance apart 0.61 d n sin = wavelength of light n=refractive index of medium between objective lens and cover slide =aparture angle (or ability of lens to gather light 21/12/54 AIT 191 Microbiology n sin = numerical apparture ที่มา Hogg (2005) 32 Usage • Assuming that the highest NA. of lens is 1.4 and the lowest visible wavelength is 400 nm • Therefore, the resolution is 0.61 400 d 0.17 mm 1.4 21/12/54 AIT 191 Microbiology 33 Magnification ที่มา http://www.microscopyu.com/articles/fo rmulas/formulasmagrange.html • Total magnification = magnification of objective lens x magnification of eyepiece • When the magnification is over the limit of resolution (limit of human eye), it will give empty magnification • Useful magnification is approximately 1000 x NA 21/12/54 AIT 191 Microbiology 34 Light and medium • The ability of a microscope objective to capture deviated light rays from a specimen is dependent upon both the numerical aperture and the medium through which the light travels. ที่มา http://www.olympusmicro.com/primer/anatomy/immersion.html 21/12/54 AIT 191 Microbiology 35 ที่มา http://www.olympusmicro.com/primer/anatomy/numaperture .html 21/12/54 AIT 191 Microbiology 36 Lens, oil immerssion, refractive index and Numerical aperture 21/12/54 AIT 191 Microbiology 37 Precaution ที่มา Benson (2001) 21/12/54 Using only lint-free cleaning paper Xylene Clean lens every time after used Always start from lower magnification Use oil immersion with 100x lens AIT 191 Microbiology 38 Stereo microscope • Use the same principle as Bright field microscope • With lower magnification (200x) • Mostly use for examining surface / 3 dimension ที่มา ที่มา http://microscopetalk.files.wordpress.com/2010/07/monilinia_fructico http://www.microscopyu.com/articles/stereomicroscopy/stereointr 21/12/54 39 AIT 191 Microbiology la_sporangium_stereo2.jpg o.html 21/12/54 AIT 191 Microbiology 40 Microscopes • • • • • Bright field Phase contrast Dark field Fluorescence Electron microscope – SEM – TEM 21/12/54 AIT 191 Microbiology 41 Optical microscope • Light • Lens • Bright field, Phase contrast, Dark field Bright field 21/12/54 Dark field AIT 191 Microbiology phase contrast42 Bright field • Normal compound light microscope • Use for observing microorganisms • Microorganisms are needed to be stained to increase contrast between background and cells • If not use other kinds of microscope such as dark field or phase contrast microscope 21/12/54 AIT 191 Microbiology 43 Dark field microscope • A darkfield microscope is used to examined lived microorganisms that cannot be seen by ordinary light microscope method. • A darkfield microscope uses a darkfield condenser that contains an opaque disk. The disk blocks light that would enter the objective lens directly. • Only light that is reflected off (turned away from) the specimen enters the objective lens. Because there is no direct background light, the specimen appears light against a black background-the dark field 21/12/54 AIT 191 Microbiology 44 Phase contrast microscope • Phase contrast can be used to examine internal structure of lived microorganisms. • In phase-contrast microscopy, the specimen is illuminated by light passing through an annular (ring shaped) diaphragm. Direct light rays (unaltered by the specimen) travel a different path than light rays that are reflected or diffracted as they pass through the specimen. These two sets of rays are combined at the eye. Reflected or diffracted light rays are indicated in blue; direct rays are red. (Bottom) Phase-contrast microscopy shows greater differentiation of internal structures and clearly shows the pell icle. 21/12/54 AIT 191 Microbiology 45 Fluorescence microscope • Fluorescence: a phomonenone that substance absorb a short wavelength of light and giving off a longer wavelength of light. • Some microorganisms fluorescence naturally. • Some is not. They are needed to be stained with fluorophore or fluorochrome 21/12/54 AIT 191 Microbiology 46 Fluorescence microscope • Fluorescence antibody technique • Create antigen (foreign object) into body and let body manufacture antibody| remove and extract antibody • Similar to bright field but different at light source (mostly UV) and wavelength selection device 21/12/54 AIT 191 Microbiology 47 Confocal microscope • This is another type of light microscope (fluorescence one) • But instead of illuminating the entire field, in confocal microscopy, one plane of a small region of a specimen is illuminated with a shortwavelength (blue) light which passes the returned light through an aperture aligned with the illuminated region. • However, it can be used to reconstruct 3D of cells 21/12/54 AIT 191 Microbiology 48 Electron microscope • Shorter wavelength of electron high resolving power • SEM & TEM 21/12/54 AIT 191 Microbiology 49 EM • It is suitable for examine object smaller than 0.2 mm such as internal organ of microorganism 21/12/54 AIT 191 Microbiology 50 New Tool of Fluoresence Microscope • Super Resolution Fluorescence Microscope Microscopy • Probe Microscopy – Magnifies more than 100,000,000 times – Two types • Scanning tunneling microscopes • Atomic force microscopes Probe microscopy Figure 4.14 Staining • Increases contrast and resolution by coloring specimens with stains/dyes • Smear of microorganisms (thin film) made prior to staining • Microbiological stains contain chromophore • Acidic dyes stain alkaline structures; more commonly, basic dyes stain acidic structures Preparing a specimen for staining Figure 4.15 Staining • Simple stains • Differential stains – Gram stain – Acid-fast stain – Endospore stain • Special stains – Negative (capsule) stain – Flagellar stain Simple stains Figure 4.16 The Gram staining procedure Figure 4.17 Ziehl-Neelsen acid-fast stain Figure 4.18 Schaeffer-Fulton endospore stain Figure 4.19 Negative (capsule) stain Figure 4.20 Flagellar stain Figure 4.21 DIRECT COUNT Direct Count • Through microscope • Using special equipment “Haemacytometer” • Counting number of microorganism per field or view under microscope • If the volume of sample is known, cell concentration of microorganism can be calculated. • However, it is hard to distinguish between live and death microorganism. http://www.fisheries.go.th/cf-chan/plankton/hema/hema.htm http://www.fisheries.go.th/cf-chan/plankton/hema/hema.htm PLATE COUNT METHOD Plate Count Method • Indirect method • Using culturing medium for growing inoculation cells • Count only viable cell as colony forming unit (bacteria) or CFU • Need aseptic techniques • 2 sub categories – Spread plate – Pour plate Problems • • • • • Sometime Too numereous to count (TNTC) Serial dilution | mostly 10 fold dilution 1 mL (sample) + 9 mL (sterilie buffer/water) If dilute too much Too few to count (TFTC) Standard ~ 30 – 300 CFU Calculation • If you found 47 CFU on petri dish of dilution at 10-5 • How many CFU/mL of original sample? • The answer is 47 x 105 or 4.7 x 106 Example • 5 g of contaminated shrimp is tested for bacterial count using plate count method. 5 g of shrimp is mixed with 100 mL of sterile buffer. Then, the mixture is diluted (10 fold serial dilution) to 10-4 and spread on culturing plate using 0.1 mL of the mixture. The result is shown as followed. How many cells of bacteria contaminate shrimp in CFU/g? Dilution CFU 10-1 TNTC 10-2 TNTC 10-3 134 10-4 17 answer • (134 x 10 x 104 x 100)/5 • 26.8 x 107 • 2.68 x 108 Spread vs Pour plate technique Spread plate • Aerobic • 0.1 mL • Using spreader to distribute bacteria on prepared plate • Plate can be prepared beforehand (before spreading) • Serially diluted • 30-300 CFU (count only colony on surface) Pour plate • Aerobic and Anaerobic • 1 mL • Swirling to distribute bacteria • Serially diluted • 30-300 CFU (count both colony on surface and inside midium) Turbidimetry method • • • • Indirect measurement of bacterial cells concentration Measure both live and death cells Fast at long run Need to do a calibration curve between bacterial cell count and absorbance • Suitable for only microbial in liquid form TURBIDIMETRIC METHOD Wavelength ~ 420-660 nm Must set blank with sterile Broth Cell count must be greater than 107 CFU/mL Turbidimetric method • If the OD deviates from a straight line, sample must be diluted with sterile culture media. • This technique can be used to rapidly quantify bacteria cell during fermentation. • However, limitation of this method is opacity of fermentation medium such as milk. FILTRATION This method can be used to quantify coliform in wate Cell dry mass (filamentus fungi, yeast) The cell mass of a very dense cell suspension can be determined by this technique. In this technique, the microorganisms are removed from the medium by filtration and the microorganisms on filters are washed to remove all extraneous matter, and dried in dessicator by putting in weighing bottle (previously weighted). The dried microbial content is then weighted accurately. This technique is especially useful for measuring the growth of microfungi. It is time consuming and not very sensitive. Since bacteria weigh so little, it becomes necessary to centrifuge several hundred millions of culture to find out a sufficient quantity to weigh. การแยกเชื้อจุลินทรี ย ์ การเลี้ยงเชื้อจุลินทรี ย ์ เลี้ยงในสภาพเหมาะสมกับจุลินทรี ยท์ ี่ตอ้ งการศึกษา • อาหารเลี ้ยงเชื ้อ – เหลว/แข็ง • อุณหภูมิ • ออกซิเจน • pH 21/12/54 AIT 191 Microbiology 91 Media Types Media Complex Defined Purpose Grow most heterotrophic organisms Grow specific heterotrophs and are often mandatory for chemoautotrophs, photoautotrophs and for microbiological assays Selective Suppress unwanted microbes, or encourage desired microbes Differential Distinguish colonies of specific microbes from others Enrichment Simlar to selective media but designed to increase the numbers of desired microorganisms to a detectable level without stimulating the rest of the bacterial population Reducing Growth of obligate anaerobes http://www.sigmaaldrich.com/analytical-chromatography/microbiology/learning-center/theory/mediapreparation.html 21/12/54 AIT 191 Microbiology 92 Media Types • General-Purpose medium: The medium that is design to grown large spectrum of microorganisms (nonsynthetic and contain a mixture of nutrients) – Examples include nutrient agar and broth, brain-heart infusion, and trypticase soy agar (TSA). TSA contains partially digested milk protein (casein), soybean digest,NaCl, and agar. • Enriched medium: An enriched medium contains complex organic substances such as blood, serum, hemoglobin, or special growth factors (specific vitamins, amino acids) that certain species must have in order to grow. – Blood agar 21/12/54 AIT 191 Microbiology 93 • Selective Medium: A selective medium contains one or more agents that inhibit the growth of a certain microbe others (D) and thereby encourages, or selects, microbe D 21/12/54 AIT 191 Microbiology 94 • Mannitol salt agar (MSA) contains a concentration of NaCl (7.5%) that is quite inhibitory to most genus Staphylococcus, which grows well in this medium amplified in very mixed samples. • Bile salts, a component of feces, inhibit mostgrampositive bacteria while permitting many gram-negative isolating intestinal pathogens (MacConkey agar, eosin contain bile salts as a selectiveagent. • Dyes such as methylene blue and crystal violet also inhibit certain gram-positive bacteria. Other agents that antimicrobic drugs and acid. Some selective media to favor the growth of a pathogen that would otherwise low numbers in a specimen. Selenite and brilliant isolate Salmonella from feces, and sodium azide is used water and food. 21/12/54 AIT 191 Microbiology 95 • Differential Medium: Differential medium grow several types of microorganisms and are designed to display visible differences among those microorganisms. Differentiation shows up as variations in colony size or color, in media color changes, or in the formation of gas bubbles and precipitates 21/12/54 AIT 191 Microbiology 96 • For example, • MacConkey agar contains neutral red, a dye that is yellow when neutral and pink or yellow when neutral and pink or red when acidic. A common intestinal bacterium such as Escherichia coli that gives off acid when it metabolizes the lactose in the medium develops red to pink colonies, and one like Salmonella that does not give off acid remains its natural color (off-white). • Spirit blue agar is used to detect the hydrolysis (digestion) of fats by lipase enzyme. (digestion) of fats by lipase enzyme. Positive hydrolysis is indicated by the dark blue color that develops in colonies 21/12/54 AIT 191 Microbiology 97 • Reducing medium: A reducing medium contains a substance (thioglycollic acid or cystine) that absorbs of oxygen in a medium, thus reducing its availability. 21/12/54 AIT 191 Microbiology 98