Microbiology Lab 3: Aseptic Technique PDF

MICROBIOLOGY LAB LABORATORY 3: ASEPTIC TECHNIQUE ASEPTIC TECHNIQUE MEDIA Aseptic Technique Media Was developed in the early to mid-1800s Refers to substances or mixtures used to through the effo...

MICROBIOLOGY LAB LABORATORY 3: ASEPTIC TECHNIQUE ASEPTIC TECHNIQUE MEDIA Aseptic Technique Media Was developed in the early to mid-1800s Refers to substances or mixtures used to through the efforts of such scientists: grow and cultivate microorganisms in a ○ Robert Koch laboratory setting such as: ○ Louis Pasteur ○ Bacteria ○ Ignaz Semmelweis ○ Fungi ○ Joseph lister ○ Viruses Has been modified over time as more ○ Protists knowledge was gained within the field These provide necessary nutrients, pH of microbiology and culturing balance, and other conditions that methodologies advanced. support the growth of microorganisms. Has 3 essential goals: Growth Media Can be classified into 2 ○ Prevent contamination main types: (unwanted microbial growth) of ○ Solid Media specimens or culture. Contain a solidifying ○ Prevent contamination of self. agent, typically agar ○ Prevent contamination of the derived from seaweed work area (lab space). (red algae), which Good aseptic technique: no solidifies at room contamination temperature and forms a gel-like substance. Semi-solid. Gelatin like This allows microorganisms to grow on the surface of the medium as distinct colonies. Examples: Nutrient agar Blood agar MICROBIOLOGY LAB LABORATORY 3: ASEPTIC TECHNIQUE Potato dextrose cannot be determined agar only that an organism is motile. ○ Liquid Media ○ Plates Do not contain a Isolation of colonies solidifying agent and Use of petri plates remain in a liquid state. ○ Slants These media are often Long-term storage used when a large (several weeks – a few quantity of months) of cultures. microorganisms is Bacterial cells will have different needed, or when characteristics when grown on solid studying versus liquid media. microorganisms in Media can also be further classified suspension. based on their composition and Examples: purpose: Nutrient broth ○ Nutrient Media Tryptic soy Contain a variety of broth nutrients such as carbs, 4 Basic Types: proteins, vitamins, and ○ Broth minerals that support Determining growth the growth of a wide characteristics in a range of liquid culture and microorganisms. growing many cells in a ○ Selective Media small space. Are designed to ○ Deep selectively promote the Determining motility growth of specific types and oxygen usage of a of microorganisms culture. It should be while inhibiting the noted that the growth of others. mechanism of motility MICROBIOLOGY LAB LABORATORY 3: ASEPTIC TECHNIQUE Incorporating specific test tube is opened and inhibitors or selective compared to plates. agents. Lack sufficient surface Only 1 microbe area to achieve isolation allowed. in most cases. ○ Differential Media Plates Allow for the Larger surface area differentiation of Easiest form of media microorganisms based to contaminate, it is on specific biochemical important to hold a or physiological plate in such a way as to characteristics. minimize air exposure. Often contain indicators Can be used to obtain or substrates that isolated colonies. produce visible changes Deeps in color or appearance Solid form of media in response to certain Lack the ability to metabolic activities of provide isolated the microorganisms. colonies because growth only occurs COLONIES along a narrow Colonies inoculation band ❖ Are formed only on solid media where termed the stab line or cells can form visible masses on top of slightly away from the agar surfaces. stab line in motile ❖ Slants and plates differ in their surface species. area Contain an oxygen Slants gradient where oxygen Will have a smaller is plentiful at the top at surface area and a the deep but limited at smaller exposure to the the bottom of the deep environment when the due to poor oxygen MICROBIOLOGY LAB LABORATORY 3: ASEPTIC TECHNIQUE diffusion across thick agars. ❖ Broths Growth is indicated by a general cloudiness called turbidity. Cells may form clumps termed flocculent that are suspended within the turbid broth. Oxygen-loving cells may cluster at the top of an inoculated broth and form a film on the broth surface termed a pellicle. Cells grown to saturation – that is cells that fall out of solution and settle in a precipitate at the bottom of the tube – are referred to as sediment. Sediment can usually be resuspended by lightly thumping the tube to remix the culture. MICROBIOLOGY LAB LABORATORY 3: ASEPTIC TECHNIQUE OTHER NOTES: Nutrient Agar/Media ❖ Colony ○ For general purpose agar Visible masses on top of agar. ○ For any microbes Only seen on solid media (plate, ○ Contains beef extract, peptone, slant, deep) and agar. Hemolysis ○ Examples: Is the result of the lysis Potato-Dextrose Agar of blood cells that are For cultivating components of blood fungi (yeasts agar. and molds) TYPES OF MICROBIOLOGICAL MEDIA Selective Agar/Media ➔ Agar Deep (stabbing plate method) ○ These media contain additives ◆ Let stand with solidify that inhibit the growth of certain ◆ Inoculating loop half of the deep microorganisms while allowing ◆ Non-motile the growth of others. (do not move away ○ Examples: from stab line) MacConkey Agar ◆ Motile For (move away from stab gram-negative line) bacteria ◆ Needs inoculating needle and Blood Agar test tube Differential Media/Agar ➔ Agar Slant ○ Allow for the differentiation of ◆ Test tube and inoculating loop microorganisms based on their biochemical properties or other characteristics. ○ Examples: Blood agar hemolytic abilities MICROBIOLOGY LAB LABORATORY 3: ASEPTIC TECHNIQUE MEDIA CONTAMINATION ➔ Agar Plate Pure Culture ◆ Streak Plate/Agar Plate ○ Refers to a population of cells Uses inoculating loop or microorganisms that Air-exposure; aerial originates from a single monitoring microbial species. ◆ Swabbing Plate ○ Contains only one species of Uses cotton swab organism. ◆ Pour Plate/Serial Dilution Mixed Culture Uses pipette or flask ○ Consists of multiple different Inoculating empty petri species of microorganisms dish plate growing together in the same ◆ Spread Plate environment. Use L-shaped rod ○ Colonies have different sizes Serial Dilution and shape - The purpose is to estimate the ○ Subculture of colonies through concentration of a sample inoculating loop - Used to decrease a bacterial Contamination concentration to a required ○ Different form of colonies concentration for a specific test method, ○ Do not mix or blend or to a concentration which is easier to ○ Entire substrate count when plated on agar plate. - Population count MICROBIOLOGY LAB LABORATORY 3: ASEPTIC TECHNIQUE Culture Media MAINTAINING CULTURE PURITY - Contains nutrients, growth promoting Sterilization factors, energy sources, buffer salts, ○ Is the complete removal all minerals, metals, and gelling agents. vegetative cells, endospores, - Culture medium: nutrients prepared for and viruses from an item microbial growth Vegetative - Sterile: no living microbes actively - inoculum: introduction of microbes into dividing medium Cannot be - Culture: microbes growing in/on killed culture medium Bacteria GELLING AGENTS Salmonella Agar Killed by ○ Complex polysaccharide autoclave ○ Used as a solidifying agent for Endospores culture media in Petri plates, Bacillus spp slants, and deeps. Clostridium ○ Generally not metabolized by (food poison) microbes. Autoclave ○ Liquefies at 100 C ○ Uses moist heat (steam) under ○ Solidifies at 40 C pressure to destroy all life ASEPTIC TECHNIQUE forms. ◦ Disinfection 45 angle until red hot Cool down for 15 secs ○ Is the killing or growth inhibition of vegetative microbes. ○ Examples: Bleaching agent Zonrox Always mix with water MICROBIOLOGY LAB LABORATORY 3: ASEPTIC TECHNIQUE Hydrogen peroxide (for Allows organisms to killing; inhibiting) grow under optimal Antiseptics conditions. ○ Are antimicrobial chemicals Temperature with or safe for use on living skin or without oxygen. tissues. Mesophilic ○ 70% for skin Organism that ○ 90% for glasswares grows best in CULTURING MICROBES moderate Five I’s temperature. ○ Inoculation Room temp Producing a pure Aerobic culture With oxygen Introduce bacteria into a Gaspak growth medium using Is a method aseptic technique to used in the prevent contamination production of Tools: an anaerobic Bunsen burner environment. Loop Candle jar needle Reduces ○ Isolation oxygen Colony on media, one ○ Inspection kind of microbe, pure Observation of culture characteristics (data) Isolation on general and Colony morphology, special (differential microscopic media) examination (gram’s ○ Incubation stain) Growing microbes Systematic recording of under proper conditions DATA MICROBIOLOGY LAB LABORATORY 3: ASEPTIC TECHNIQUE ○ Identification General Growth Media Use of data, correlation, Nutrient Agar to ID organisms to exact Trypticase soy agar species. Differential Media Resources: flow charts, MacConkey Agar Bergey’s manual Ethylene-Methylene Blue Example: Shigella Salmonella Agar Gram - bacilli, These have dyes, salts, inhibiting agents ferments lactose, green sheen on EMB (E. coli) Gram + cocci, grape like clusters, golden yellow colonies, catalase + coagulase + resistant to Methicilin (MRSA) (S. aureus) NOTE: - Petri dish lid is opened as little as possible angled and kept over the base. - Each petri dish holds about 20 mL.