Practicum I SG Micro [720] PDF

Preview

Summary

practicum...

Description

Microbiology Study Guide Practicum I Microscope  When we get it, uncover it and put the cover away, then plug it in, turn on the light switch.  On the side you see the light knob that is the light intensity, sometimes we use two light dots with certain strains but never 3,4, or 5.  Arm/Neck: This is not part of the optical path of the microscope but is the mechanical support that holds the upper part in place.  Ocular Lens: On the top is the ocular lens, this is the eyepiece, and it is 10x magnification. Adjustable for eyes, and adjusts to your eyes each time. You want to be able to see the pointer.  Objective Lenses: In the middle is the cluster of objective lenses with a revolving nosepiece. It has four different objective lenses. -(Shortest) Red 4x (use for observing slides)- Scanning -Yellow 10x -Blue 40x -(Longest) White 100x  Stage: Black Platform, holds slides and usually has a hole to allow light to pass through and enter lenses, has a slider to move the hook that holes the slide glass onto the stage  Knobs on Stage: Two knobs on bottom the top one goes forward and back, and the bottom one is left and right  Diaphragm: This is the clippie thingy under the stage that has a 100x, 40x, 10x on it, this is the diaphragm, the regulates the amount of light going to the condenser, should match the objective lens you are on  Condenser: Focuses light onto the specimen, collects and concentrates the light up to the object  Light: Source comes from the bottom

 Course Adjustment Knob: To focus you look through the ocular lens and focus with big knob on the sides it goes up and down fast, stage moves so you can get initial focusing  Fine adjustment Knob: This is for fine focusing of up and down, smaller part on big knob  Names For Shapes  Cocci (Sphere)  Diplococcus (2 Spheres), Streptococcus (String of Spheres), Staphylococcus (Cluster of Spheres), Tetrad (4 spheres)  Bacilli (Rod),  Diplobacillus (2 rods), Streptobacillus (String of rods)  Spirochetes (Very tight spiral)  Spirilla (Less of a spiral)  Vibrio (Bean shaped) Using the Microscope  4x focus, turn the revolving nosepiece ONLY to go to 10x  The microscope is par focal so the objectives will stay in focus when the magnification is changed  Before 100x you need to put 40x and 100x in the sides of the slide and put a drop of immersion oil without a coverslip, then go to 100x.  The total magnification will now be 1000x.  When done remove the slide in 4x position, do not have to lower the slide just to 4x then back up to 100x with oil.  When all done clean the 100x lens with optical lens wipes not the ones in the green box those will scratch the lens, green box (KIMTECH) ones can be used for stuff without the lens  Turn the light off, unplug, and store, all slides are tossed in biohazard

4 different Bacterial Stains on a plate 1. Micrococcus luteus -

In like tap water, skin, soil, it is a coccus Gram positive bacteria that forms in tetrad which is 4 coccus together (NonPatho)

2. Bacillus subtilis - From the soil, Rod, Gram positive bacteria (NonPatho) 3. Escherichia coli -A rod shape, smaller than Bacillus Subtilis Gram Negative bacteria , sometimes when stained it looks like a coccus 4. Staphylococcus aureus -Coccus, Gram positive, Staph means clusters of coccus, (Patho)

Staining Techniques

Negative Stain Bacillus subtilis

Negative stain Micrococcus luteus

-

1) Negative Stain: Two different names, the background stain or the indirect stain.

-

So it stains the background of the cell and not the cell itself

-

Uses acidic dyes (Negatively charged), that will remain outside the cell because the bacterial cell surface is negatively charged, so when you use the acidic dye that is also negatively charged it will repel it, that is why it is called indirect staining because it is not really staining

 4 Acidic Stains used in Negative Stains (N.I.C.E.)

 Remember they are acidic (Acids are NICE)  1. Nigrosin (we use this)  2. India Ink  3. Congo Red  4. Eosin  Procedure for Negative Staining: 1. You have a plate, a loop and a slide glass. 2. Turn the valve all the way up on the Bunsen burner and use a striker to light the burner (ice cream scoop thing), open the vents or close them to get it on open usually helps 3. Put the loop in a high position to sterilize to get shiny orange without shiny color it is not sterilizing, for 10 seconds, too cool down put loop in area of plate without bacteria 4. Pick a bacteria, we are using #1 or #2 from the list (Micrococcus luteus or Bacillus subtilis) Then scrape off on the surface of your plate, don’t dig, its on the surface 5. Place On slide glass on one side call him to check, too much it’ll be too thick to see cell layers and too little there is not enough to look at 6. Add 1 Drop of Nigrosin *Acidic Dye 7. Mix until there are no chunks 8. Use Second slide glass at 45 degree angle press firmly and glide to the other side, do not stop the whole way, now it is spread evenly over the glass, toss the second glass in biohazard 9. Air dry then Ready!

Simple Stain Bacillus subtilis

Simple stain Micrococcus luteus

 2) Simple Stain: Uses basic Dyes (because when you are simple you are basic)  Basic dyes are positively charged, so the negative cell surface and the positive basic dyes attract and it dyes the cell itself, it goes into the cell and stains it

 3 Basic Dyes Used in Simple Stain (MCS)  1. Methylene Blue (Blue) (2MIN)  2. Crystal Violet (Violet) (30SEC) (Most pop because of short wait time)  3. Safranin (Red) (2MIN)  Simple Stain Procedure  First part is called BACTERIAL SMEAR 1. Must do this before staining to mount the stain 2. Put the bacteria right in middle 3. Drop H2O next to it on the side from the squeeze bottle and use the loop to bring H2O to the bacterial cell 4. Not too much or you cant see the bacteria, use tissue if too much water 5. Mix and spread all over glass, wider you spread the faster it dries the better chance you have something good to look at 6. (Remember every time you change bacteria to sterilize the loop) 7. Air dry

8. Heat Fix, if you don’t do this the stain will run off the slide, pass over the flame a few times and can use tweezers if you want to. 9. Now Stain it with staining pan, on the staining pan 10. Flood the slide glass with your choice of basic dye and make it cover the whole area then wait and remember the times for everything 11. Rinse off basic dye with squeeze bottle (eye water) then blot the slide glass dry, the bottom you can wipe with KIM wipes but be gentle on the top 12. Then ready to look at! Use same bacterial species as negative stain.

Gram Stain Escherichia coli and Staphylococcus aureus  3) Gram Stain: This is also called differential stain, it gives you two colors like gram positive or gram negative  Procedures for Gram Stain  First part is BACTERIAL SMEAR 1. Must do this before staining to mount the stain 2. Put the bacteria right in middle (Using two so similar amount of both) 3. Drop H2O next to it on the side from the squeeze bottle and use the loop to bring H2O to the bacterial cell

4. Not too much or you cant see the bacteria, use tissue if too much water 5. Mix and spread all over glass, wider you spread the faster it dries the better chance you have something good to look at 6. (Remember every time you change bacteria to sterilize the loop) 7. Air dry 8. Heat Fix, if you don’t do this the stain will run off the slide, pass over the flame a few times and can use tweezers if you want to  We are using #3 and #4 species on the list (Escherichia coli (gram – (Pink)) and Staphylococcus aureus (Gram + (Purple))  So if we mix them we can see the purple and pink together  Then we will stain in the pan 1. Crystal Violet (Basic Dye): This is the primary stain (30SEC), then rinse off with water, NO BLOTTING 2. Grams Iodine: This is a mordant (Does not give off its own color it only adheres to #1) (1MIN), then rinse off 3. 95% Ethanol: This is the decolorizer, (30SEC ONLY) then rinse off 4. Safranin (Basic Dye): This is the counter stain (2MIN), rinse off, BLOT dry  Done! Gram + (S.A) After Step 1: Purple After Step 2: More purple After Step 3: Purple (purple color locked in

Gram – (E.C) Purple Purple No color (Lose color due to thin

by dehydration with the G+ layer) After Step 4: Purple (No more room to

peptidoglycan layer) Red/Pink (A lot of room to stain so it takes

stain)

the pink)

Acid Fast Stain Mycobacterium smegmatis and Escherichia coli  4) Acid Fast Stain: This is a differential stain so you will see two different colors when the staining is done, done mainly for the genus Mycobacterium  For Mycobacterium, the cell wall is a mycolic acid (lipid), we cant use simple stain or gram stain because of it This is why we use the HOT PLATE, the hot plate breaks up the mycolic acid so we can stain it  Types of Mycobacterium for Acid Fast Stain 1) Mycobacterium tuberculosis (TB) (Patho) 2) Mycobacterium leprae (Leprosy) (Patho) 3) Mycobacterium Smegmatis (NonPatho, Rod, Gram Positive) *Use this in lab*  Types of Things used with Mycobacterium Smegmatis For Acid Fast (Differential) Stain 1) Escherichia coli (-) Rod 2) Bacillus subtilis (+) Rod 3) Klebsiella pneumonia (-) (red)  Procedure for Acid Fast Stain:  First part is BACTERIAL SMEAR 1. Must do this before staining to mount the stain 2. Put the bacteria right in middle (2 so same amount for both)

3. Drop H2O next to it on the side from the squeeze bottle and use the loop to bring H2O to the bacterial cell 4. Not too much or you cant see the bacteria, use tissue if too much water 5. Mix and spread all over glass, wider you spread the faster it dries the better chance you have something good to look at 6. (Remember every time you change bacteria to sterilize the loop) 7. Air dry 8. Heat Fix, if you don’t do this the stain will run off the slide, pass over the flame a few times and can use tweezers if you want to  Then take a hot plate, and leave the right knob that is for magnetic stuff, the left is the temp control turn Clockwise to 80-85 degrees Celsius, when it flashes its working when its solid its done  Put the slide glass on the hot plate and put filter paper right on it, add stain and flood the filter paper with Carbolfuchsin for 5 minutes (Primary Stain, makes both reddish)  It needs to stay wet, and keep adding time to time, remove air bubbles if any and use alcohol to clean the plate after  Peel off the filter paper, toss it  Put slide glass in staining pan, wait a minute or two to cool off then rinse off the remaining stain  Then add Acid Alcohol for 30 seconds, (kinda the decolorizer) this will lock the stain up in the Mycobacterium smegmatis, but remember Escherichia coli will lose its color because it is Gram –  Rinse off acid alcohol with H2O and apply the (counter stain) Methylene blue (2MIN), rinse off with H2O and blot dry  Ready!  Remember that Mycobacterium smegmatis is yellow with a lot of lipids, so you don’t need to use little loop, use bigger loop and squeeze when you mix it with water because it is Hydrophobic.

 Mycobacterium smegmatis should form a cluster; find the red spot on 4x to find cluster and the Blue Escherichia coli will be all over.  So at 100x the Escherichia coli will be blue (Acid fast -), and the Mycobacterium smegmatis will be red (Acid fast +)

 Lifecycle of a Spore  Vegetative State (Keeps growing without producing endospores)   Forespore (This is the prespore)   Matured (The Forespore is matured by adding more layers outside and putting more stuff inside)   Cell Lysis (Cell Lysis occurs and the endospore is released into the environment)   Free Endospore (The endospore is now free and is resistant to a lot of environments but it is dormant and not dead but there is no metabolism)   Germination (This is when it meets with nutrients and is able to multiply)

Spore Stain Bacillus subtilis  5) Spore Stain: This is from the endospores, two most well known are Bacillus and Clostridium, they are both gram + bacteria that produce these two well known endospores  We used Bacillus subtilis in lab for this

 Procedure for Spore Stain  First part is BACTERIAL SMEAR 1. Must do this before staining to mount the stain 2. Put the bacteria right in middle (2 so same amount for both) 3. Drop H2O next to it on the side from the squeeze bottle and use the loop to bring H2O to the bacterial cell 4. Not too much or you cant see the bacteria, use tissue if too much water 5. Mix and spread all over glass, wider you spread the faster it dries the better chance you have something good to look at 6. (Remember every time you change bacteria to sterilize the loop) 7. Air dry 8. Heat Fix, if you don’t do this the stain will run off the slide, pass over the flame a few times and can use tweezers if you want to  Put slide glass on hot plate, get filter paper and flood with Malachite green (Basic dye) for (5MIN)  This will penetrate in the layers and dye the cell  Put the slide glass in the pan and wait 2 minutes to cool then rinse off green in pan with H2O  Then apply Safranin (2MIN), rinse off with H2O and blot dry, then its ready  Endospores is the Christmas tree stain so you will see the green endospore from the Malachite green and the Red cytoplasm from the Safranin, sometimes just one color but you want to find both.

Capsule Stain with Klebsiella pneumoniae  6) Capsule Stain: Remember for some cells there is the glycocalyx that some bacterial species have either as a Capsule (Thick Tight) or a Slime Layer (Thin Loose), the capsule is mainly sugar.  We use Klebsiella pneumoniae (Gram -), rod shaped (Pathogenic), this is the number 1 bacterial pneumonia, some strains are antibiotic resistant  This Klebsiella pneumoniae will be given to use on a red plate, and it is very slimy because of the capsules (He grew this on MacConkey Agar)  MacConkey agar is used because a lactose component inside it promotes more capsule production to see the capsule stain  Capsule stain has NO BACTERIAL SMEAR, unique procedure  Capsule Stain Procedure 1. We will negative stain the Klebsiella pneumoniae 2. Sterilize Loop 3. Then scrape bacteria off plate with loop 4. Place On slide glass on one side, too much itll be too thick to see cell layers and too little there is not enough to look at 5. Add 1 Drop of Nigrosin *Acidic Dye 6. Mix until there are no chunks 7. Use Second slide glass at 45 degree angle press firmly and glide to the other side, do not stop the whole way, now it is spread evenly over the glass, toss the second glass in biohazard 8. Air dry. NO HEAT FIX 9. Then put it in staining pan, flood the slide glass with Crystal Violet (30SEC) 10. NO HEAT FIX, so the Klebsiella pneumoniae is floating around in the crystal violet, and we rinse it off with .85% Sodium Chloride (NaCl) which is a saline solution, but be careful and wash GENTLY

11. We use the saline solution to wash off the crystal violet for the capsule stain to keep the capsule integrity, as saline does this better than H2O 12. NO BLOT DRY you will make the capsules go away, but can clean back 13. Now ready!  So basically this dye, when you stain with Nigrosin it stains the outside of the cell, the background (Remember because you negative stain with Nigrosin)  Then when you stain with Crystal Violet itll be on the inside and the outside of the cells, BUT when you rinse with NaCl (saline) solution, itll make the capsule bright  With the 4x look at the dark part to get the capsule when you zoom in, The CV stain will be inside the cell, capsule should be bright white and background is Nigrosin stain  5 Sterilization Methods (Killing all living organisms) (G.U.A.D.F.)  1) Autoclave: high pressure and hot temperature to kill the germs, at about 121 degrees Celsius for 15-20 minutes at 15 PSI (pounds per square inch)  The autoclave is most popular for microbiologists  Used for mainly glass things  No plastic in it because itll melt, everything else can be autoclaved  2) Dry Heat (Oven): HIGH temps, more than the autoclave at about 160-170 degrees Celsius for more than 2 hours  Can use glass, cylinders, pipets, flasks etc.  Not any longer than 2 hours or itll distort the stuff  3) UV radiation: This is the most destructive form, the UV wavelength is 260 nm, to kill germs  This high wavelength causes mutation in DNA and the cells cant replicate and it dies  UV cannot penetrate barriers like glass, water, plastic Etc.

 This is very limited application, usually only good in little box in lab corner  4) Filtration: Every filter has a pore and it is used when one part of your substance can be heat damaged.  0.22 um is the filter size. The germs will be captured when you attach the little filter to the syringe.  5) Gas: The gas we are using is Ethylene gas  The gas molecule is small and they can penetrate plastic wrap, this essentially chokes the cell  It is explosive and unstable and binds to any protein  Types of Culture medium  Physical State 3 types  1) Liquid (Broth): We use this culture medium to obtain a large amount of cells per unit medium instead of just on top of a solid its all over in the liquid.  2) Semi Solid: In the test tube like jello, It is used in our unknown project  We stab the semi solid medium all the way to the bottom then pull it out, you can check bacterial motility to see if they spread all over and grow, if not the growth will be limited to the stabbing path  We can also use this medium to promote anaerobic growth  3) Solid: This is usually in a petri dish and can be used for three things  To obtain a pure culture (Like a single colony)  To observe the colony appearance (Is it smooth rough compact spread colored?)  To also use for storage, it is not long term in solid medium only for a few months in fridge  Ingredients 2 types

 1) Complex Medium: In this medium at least one component you do not know the exact type of chemical structure  Examples: Yeast extract, beef extract, peptons (We don’t know the exact chemical structure of these) (Heterotrophs (Get nutrients from complex organic substances))  We usually use complex ingredients  2) Chemically Defined: We know ALL the chemical structures  Examples: (NH4)2 SO4 (Ammonium Sulfate) is made from K2HPO4 (Dipotassium Phosphate) and CaCl2 (Calcium Chloride)  We know all the chemical structures and we usually use the chemically defined for autotrophs (Gets nutrients from inorganic substances like carbon dioxide) because we don’t need a lot of nutrients made for them  Pipette Usage  The 10ml are individually wrapped. They are the big ones we were using. You can see it on the side it is up to 10ml. 10ml pipette works with GREEN or RED syringe.  Then there is a 1ml pipette that is glass. This is the smaller little one. It can go all the way up to 1 ml. 1ml pipette goes to PURPLE syringe.  Make sure to touch the side not the top because it is sterilized. The cotton top on the pipettes is to prevent contamination from the top. We scroll up to get the liquid to go up or we push the top or side to release the water.  Using the pipettes, if you put 9ml H2O in a 10ml pipette technically it is going all the way up to 1. (0 at the top, 10 at the bottom) So if you want 9ml you fill to 1, 10 ml you fill to 0, 8ml you fill to 2 etc. For the little pipette if you want .9 ml then you go to .1, for 1ml you go to 0, for .8 ml you go to .2 ml  Other Suctions  Classical Suction: This one has a button on the top to suck up and you press the S (side) to keep it and the E (next to it) to release it

 Gun Suction: The top button gets the liquid and the bottom releases it, but its too fast of a suction so we don’t use it in class.

 Labeling Plates  Your name or initials  Bacteria Name  Todays Date  Plating method  Dilution if you have it  Remember to keep the label side up so there is no condensation that will drip down if you want it to dry, we put the l...