Lab 6_Slide Set - Bacterial Transfer and Isolation_Micro50_LMC_F24 2.pdf

1. Aseptic Technique: media inoculation 2. Colony Isolation Streak for Isolation Dilution Spread Plate and Pour Plate Techniques  Aseptic technique Microbiology Terms: inoculate – to intentionally introduce onto nutrient agar or broth contaminant – gr...

1. Aseptic Technique: media inoculation 2. Colony Isolation Streak for Isolation Dilution Spread Plate and Pour Plate Techniques  Aseptic technique Microbiology Terms: inoculate – to intentionally introduce onto nutrient agar or broth contaminant – growth of unwanted microbes aseptic technique – sterile technique; all media/supplies sterilized gelatinous tough compound – few microbes can degrade septic technique – non-sterile technique autoclave – steam sterilization heats H2O under pressure pressure allows temperature to increase above “boiling point” without boiling highly effective sterilization technique agar – carbohydrate extract from red algae gelatinous tough compound – few microbes can metabolize and degrade it. nutrient broth – liquid complex medium nutrient agar – solid medium adding H2O to agar causes it to solidify – solid surface used to grow microbes  Microbiology Terms: chemically defined media – a medium whose exact composition is known complex media – medium whose exact composition varies slightly batch to batch protein – supplies organic carbon, nitrogen and energy in form of meat extracts and partially digested proteins called peptides culture types – culture is “contained” growth of laboratory bacteria Liquid broth – provide large #’s of bacteria in small space; can’t distinguish color/morphologies Agar slant – agar in test tube, top surface at a slant; provides more surf. area for long term storage Agar deep – agar in test tube, top surface flat; for bacteria needing less O2, determine motility inoculating loop – non corroding metal wire loop; used to inoculate / transfer bacteria cultures inoculating needle – straight metal wire; used to inoculate agar deeps petri dish – provides large surface area to grow bacteria and examine colonies inoculate – to intentionally introduce onto nutrient agar or broth incubation period – time stored at slightly elevated temperature (37C) to increase growth rate turbidity – cloudy broth solution from growth colony – population of cells on agar plate that arise from a single (or few) cells colony forming unit (cfu) – the cell or cells that generate the colony Inoculating different media – broth, slant, deep Media types: broth, slant, deep slant to broth inoculation  o inoculate agar slant as follows: 1. slant – loop in dominant hand; source culture broth/slant in other o sterilize loop – remove cap (carefully) from broth/slant tube; DO NOT SET DOWN CAP o with tube at an angle pass tube through flame 3x 2. immerse “cooled” loop into bacterial broth or touch slant surface / flame culture tube / re-cap / set down culture tube 3. remove cap from sterile slant tube – flame 3x 4. gently “zigzag” loop-full of culture from back to front on surface of slant / flame tube / re-cap / set down slant o re-flame loop Inoculating a slant: zig-zag slant back to front Gently!!! o inoculate agar deep as follows: USE INOCULATING NEEDLE! 1. deep – needle in dominant hand; source culture broth/slant in other o sterilize loop – remove cap (carefully) from broth/slant tube; DO NOT SET DOWN CAP o with tube at an angle pass tube through flame 3x 2. immerse “cooled” loop into bacterial broth or onto surface of bacterial slant o flame culture tube / re-cap / set down culture tube 3. remove cap from sterile deep tube – flame 3x 4. single stab to bottom with inoculating needle / flame tube / re-cap / set down slant o Re-flame loop Inoculating a deep: use inoculating needle single stab to bottom Inoculating different media  Methods for isolating colonies: streak plate technique o bacteria “streaked” with loop on plate to spread and separate/isolate colonies spread plate technique o series of culture dilutions made – volume of each “spread” over agar plate uses a spreading rod or “hockey stick” to spread plate evenly pour plate technique o volume of each dilution is mixed with melted agar and poured into petri dish cfu/mL: # of colonies / dilution factor * vol. plated  isolating bacteria by dilution techniques 1. Streak Plate for Isolation 2. Pour Plate Purpose: to isolate single colonies by spreading culture across agar plate Colony: circle of bacterial growth on an agar plate.  Mixed Colony: more than 1 species within colony  Pure Colony: colony is single species Colony Forming Unit (cfu): from 1 cell or single colonies – GENETICALLY IDENTICLE Streaking for Isolation: streaking for isolation streaking for isolation creating four “zones” zone 4 - isolated colonies  streaking for isolation Streaking for Isolation creating four “zones” Details: 1 agar plate only mixed bacteria culture Streaking for Isolation Procedure: sterilize and cool loop BETWEEN EACH STREAK 1. spread bacteria thoroughly in quad. 1 of a TSA plate 2. pass quad. #1 2-3 times and spread bacteria into quad. 2 3. pass quad. #2 2-3 times and spread bacteria into quad. 3 4. pass quad. #3 2-3 times and spread bacteria into quad. 4 Important: sterilize between EVERY STEP rotate plate slightly after completion of each “zone” streaking for isolation quadrants 2-4 use a rapid “zig-zag” motion zone 4 - isolated colonies Dilution Spread Plate Technique – Serially dilute bacteria and plate each by spreading specific volume Pour Plate Procedure 1. Serially dilute bacteria 2. Add each dilution to individual melted agar tubes 3. Pour each tube on top of individual agar plates Dilution/Pour Plate Procedure  Exercise 10 day 2 – isolating bacteria by dilution techniques(GROUPS) 1. Streak Plate for Isolation 2. Dilution Pour Plate Results: Pour Plate 1. Count colonies >250: TNTC