B=CORRECT SMEAR THICKNESS "AFTER" DRYING!
A B C
Horrible Micro SINNER - AGAR CHUNKING = SENT HOME 2ND TIME!
To Start your unknown cards begin with the Procedure Cards - (purple cards - see the complete 33 procedure card list go to BONUS POINTS in the menu list on the right or click here: Unknown Card LIST. This is where you get the information to use in putting the cards in your own words. NEXT, click on CHECK YOUR CARDS to compare your card against a good set of cards). The cards need to be alphabetical in your binder or ring OR you may use an INDEX card placing the index in alphabetical order with the page number listed to the right of that Title.
Please begin your FIRST CARD with a LAST name, first name, COLLEGE & class section #, ID#, cell number, scopre/seat number, email address etc. The SECOND CARD should be the INDEX card if the binder is NOT alphabetical. The THIRD CARD should be a SAFETY in the LAB Card.
LP1 REQUIRED First 11 typed CARDS: To BEGIN the Lab Practical 1 Exam Laboratory Exercises: click here (or on the first yellow link below this paragraph) and complete the cards you need for the first three weeks of class and lab. The cards you need to make first are: (1) Properly Washing a Slide - new card, make from video (2) Preparation of a Smear from Agar (3) Preparation of a Smear from Broth (4) Simple Staining (5) Focusing the Microscope Under 100x - new card, make from lecture and video (6) Cleaning and Storage of the Microscope - new card, make from lecture and video (7) The Negative Stain (?) (8) The Gram Stain (9) The Acid Fast Stain (10) The Endospore Stain & (11.) the Safety Card.
CHECK YOUR CARDS AGAINST MINE BEFORE YOU TYPE THEM! Have you got too many "words?" Is the FONT BIG and DARK ENOUGH TO READ from a distance? LOOK AT MINE! Where to look? Go the right hand menu item: (2) MT/LP2 then look at the second menu on the right that says CHECK YOUR CARDS. Or use this: Click here for a link to "CHECK YOUR CARDS against a good set" Link
Please begin your FIRST CARD with a LAST name, first name, COLLEGE & class section #, ID#, cell number, scopre/seat number, email address etc. The SECOND CARD should be the INDEX card if the binder is NOT alphabetical. The THIRD CARD should be a SAFETY in the LAB Card.
LP1 REQUIRED First 11 typed CARDS: To BEGIN the Lab Practical 1 Exam Laboratory Exercises: click here (or on the first yellow link below this paragraph) and complete the cards you need for the first three weeks of class and lab. The cards you need to make first are: (1) Properly Washing a Slide - new card, make from video (2) Preparation of a Smear from Agar (3) Preparation of a Smear from Broth (4) Simple Staining (5) Focusing the Microscope Under 100x - new card, make from lecture and video (6) Cleaning and Storage of the Microscope - new card, make from lecture and video (7) The Negative Stain (?) (8) The Gram Stain (9) The Acid Fast Stain (10) The Endospore Stain & (11.) the Safety Card.
CHECK YOUR CARDS AGAINST MINE BEFORE YOU TYPE THEM! Have you got too many "words?" Is the FONT BIG and DARK ENOUGH TO READ from a distance? LOOK AT MINE! Where to look? Go the right hand menu item: (2) MT/LP2 then look at the second menu on the right that says CHECK YOUR CARDS. Or use this: Click here for a link to "CHECK YOUR CARDS against a good set" Link
PAGES/Examples that you will need to make your cards for the LP1 EXAM:
CARDS:
Title: SAFETY in the LAB: (my card -USE YOUR OWN WORDS!)
PURPOSE: This card is to remind us of the common safety, sterile/aseptic technique procedures, modified universal precautions, & safe discard procedures used constantly while in a Microbiology Laboratory working with bacteria and chemicals.
DAILY/GENERAL RULES PROCEDURES:
1) When you arrive (and before and after lab), place your books etc in your CHAIR, WASH your hands thoroughly (45 sec min) with soap and rinse well. Dry you hands with paper towel and use that paper towel to spread the supplied disinfectant over the table-top to be used. Allow the table-top to air dry before use.
2) NEVER lay down a cap, needle, loop, or test tube on a table top; use the wooden blocks or test tube rack. When picking-up any test tube, use the GLASS, not the cap as caps come off in micro and are never screwed tight because the microbes need air. BRIEFLY FLAME the outside of any lid and the lip of any tube before use (with the exception of foam and cotton stoppers) to maintain sterile/aseptic technique. NOTE THAT CAPS remain on the test tube or in the crook of your little finger when off the test tube as contamination occurs when they are placed on table tops. SLANTS and TEST TUBES are labeled "around the tube at the top with a Sharpee and include last name, section, date inoculated and experiment name. DO NOT LABEL TUBES DOWN THE SLANT - you cannot see "growth" with this kind of label.
3) SLANTS and any TUBES with GROWTH especially stock and backup cultures go into incubation for 12hrs only and never again... after the initial incubation they are REFRIGERATED
4) ALL procedures done in this lab should be done in our modified universal precautions which include a long cotton or cotton-blend lab coat (something below the waist) and latex or nitrile gloves... a face shield is not necessary unless you are in danger of splashes. Please do not wear gloves in the halls; remove and discard them often. When MICROBES are out in the lab all food, drink and make-up must be out of sight and long hair must be tied back.
5) If you make a spill, disinfect it and wipe it with a paper towel and discard the paper towel in the normal trash
6) If you break glass, disinfect and CALL THE INSTRUCTOR
7) If you spill a stain, wipe it with paper towel, apply ACID ALCOHOL, wait 3 seconds and wipe with paper towel - NOTIFY the INSTRUCTOR
8) PETRI DISHES are stored, incubated and kept UP-SIDE DOWN. The labeling is done around the outside edge with a black SHARPEE on the bottom of the plate - NOT THE TOP; plates are lightly taped 2x for incubation and storage. Petri Plates are overnight and then to refrigerator they are NEVER reincubated
GENERAL RULES:
Slants are checked in 2 areas (not at the either end) for Purity by Gram staining at their first use and at least every 10 days after. All tubes are returned to the refrigerator NEVER RE-INCUBATED! Mark all slant tube tops with a color or acceptable symbol on the top as all student tubes are stored together; all tubes should be labeled with your LAST name, section number and the date created. DO NOT USE YOUR UNKNOWN NUMBER!
in All TESTING is done with an inoculates that exists in Log Growth Stage for the results to be accepted. Log Growth is a tube incubated 12-18 hrs at Opt Temp. We maintain ARTIFICIAL Log Growth by incubation/subculture (inco taken from an older tube) for 12 hrs at Opt Temp and then 5C refrigeration for no more than 21 days. Working cultures can be used up to 21 days (checked regularly for Purity). Stock cultures are rotated to Working at no longer than 14 days refrigeration & new Stocks are thensubcultured and checked for Purity. Refrigeration slows but dehydrates cultures...
**All data
***All Dis DISCARDS should be made to the discard area. Discard baskets/racks should have tubes of the same size. If there is a basket, it should be kept at an “angle” to prevent discarded tubes from mixing their contents. Paper, Plastic, and Wood items that are contaminated with unknowns or reagents should be placed directly in the ORANGE-RED BIOHAZARD BAGS (you do not have to remove marks and tape). Biohazard bags should not be used at trash cans. Glass Petri Dishes and all glass items should have the tape and marks removed with fingernail polish remover before discard. Glass Dishes should be placed up-side down in the plastic bin on the second shelf of the discard table. When the Discard racks/bins/baskets are full notify the instructor IMMEDIATELY! Discarding wrong can cause the class to be shut down as it is DANGEROUS to us all!
--------------------------------------------------
Title: How to Properly Wash A Slide (my card -USE YOUR OWN WORDS!)
PURPOSE: To remove (even light) oil from slides so that a bacterial SMEAR PREP will stick during the washes required for the STAINING process
1. Obtain a slide that has NO CHIPS or CRACKS
2. Using damp or wet gloves, make a thick "paste" of Bon Ami (brand) scouring glass powder and RUB both sides of the slides thoroughly with the paste at least 2X
3. RINSE the slide well; CHECK for BEADING-UP of WATER due to oil remaining on the slide
4. To check for washing well, place the slide under a stream of free flowing water; remove from water and count to 5; check for BEADING-UP or water "withdrawing" from areas on the slide (see video); if YES re-wash 1 to 2X & RE-CHECK for BEADING-UP - do as many times as necessary
5. DRY the slide with paper towel and handle it by the edges to avoid human oil from your fingers
----------------------------------------------------------------------------------------------
Title: Focusing the Microscope under 100x: (my card -USE YOUR OWN WORDS!)
PURPOSE: To allow for the easiest and SAFEST (to protect the $ scope from damage) way to observe bacterial preps using a 100x microscope
1) Obtain your numbered scope (use your SEAT #). Carefully remove it from the storage locker (leave any cover there) and with one hand under the foot and the other on the arm, return to your seat. Check that the POWER is off (rheostat and power switch or power wheel). Plug the scope in (in front of your lab bench) and move the power to about 90%
2) Obtain a slide with 1 drop of immersion oil already applied to the circled or stained area. Using the left -hand side of the slide holder "pinch" or "squeeze" the spring and place the slide INSIDE and fully back into the slide cut-out area. DO NOT ALLOW THE SLIDE TO RIDE ABOVE THE HOLDER or at the outside edge!LOOK UNDER the stage and open the IRIS Diaphragm ring or lever to about 90%
3) Using the 2 black stacked slide adjustment knobs beneath the left-hand side of the STAGE, move your slide so that the circled or stained area with the drop of immersion oil is directly over the condenser light.
5) Using the right-hand single black CONDENSER height knob (above the right side Course Adjustment knob) carefully raise the condenser apparatus to NEARLY or almost touch the back of the slide - DO NOT PUSH THE SLIDE UP with the condenser!
6) GRASP the nose-piece ring and carefully turn it until the 10x (yellow ring) Objective "clicks" into place
7) Using either of the two large COURSE ADJUSTMENT knobs on the ARM of the scope, move the stage ALL THE WAY UP (turn the knobs toward you) - do not move too fast and DO NOT FORCE THE MOVEMENT - when it stops moving up, DO NOT FORCE IT (if you are at GCC, make sure the LOCK is OFF before doing this or you will ruin the scope!)
8) Set the eye-width binocular oculars or eyepieces so that you see not 2 but 1 large round white view through the scope when your eyes are about 2 cm (a little less than an inch) BACK from the oculars.
9) While looking through both oculars with both eyes open, turn the COURSE ADJUSTMENT knobs slowly away from you as this will move the stage DOWN from its highest position. When you see a flash, use the inner smaller FINE FOCUS knobs establish a clear view. You may want to use the Slide adjustment knobs to CENTER the item you see in the field of view for viewing under 100x because the field shrinks as you move from 10x to 100x
10) Gently grasp the nose-piece ring and carefully click the 100x Oil Immersion lens into place and using the inner smaller FINE FOCUS knobs, create a perfect view by turning first one way a few turns and then the other. To perfect your view, grasp the Iris Diaphragm lever or ring and adjust it to the best clarity and most light. Draw what you see or call the instr. NOTE: Be careful when turning the nose-piece to turn it counter-clockwise so that you do not drag the 40x through the oil or you will have to clean it after class with lens paper the same way you do the 100x.
NOTE: See Scope Care, Cleaning & Storage below for how to clean-up your scope
-----------------------------------------------------------------------
Title: Scope Care, Cleaning & Storage: (my card -USE YOUR OWN WORDS!)
PURPOSE: To properly take care of and maintain and store the microscope with particular emphasis on protection of the 100x oil immersion lens which may be ruined if oil is left longer than 36-40 hrs
1) Turn off the light with the power wheel or switch/rheostat. Unplug and wrap the cord...
2) Put the stage all the way down (ALL THE WAY Up for Biol 112) ; move the slide clip so that it does not extend past the stage or "stick out"
3) Click the lowest (4x) Scanning objective into place
4) Lower the condenser ½ turn
5) USING LENS PAPER ONLY, Clean any oil from the 40x and then 100x objectives. DOUBLE Check both the 40x (High Dry) & 100x (oil immersion) objectives. REMOVE excess oil from the stage with a paper towel
6) Remove any “stain spatters” from the scope, table or floor IMMEDIATELY with ACID ALCOHOL (let set 3 seconds) and then remove the liquid with some paper towel
7) Take your clean scope it to a POST CLASS SCOPE CHECKER for approval before placing it in the correctly numbered spot (put on a cover if one is provided)
*CAUTIONS: The microscope is ALWAYS carried using TWO HANDS; one on the base & the other on the ARM!!! To change the objective lens or the Iris Diaphragm please grasp the beveled rings or lever DO NOT GRAB THE CONDENSER!
YOU ARE WARNED with your first scope cleaning error... at the second occurrence you are penalized 5 points per incident
-----------------------------------------------------------------------------------------------------'
Prep of a Smear from Agar: video click here
Title: Preparation of a smear from AGAR (You are Diluting to get 1-100 microbes)
Title: Preparation of a smear from AGAR (You are Diluting to get 1-100 microbes)
PURPOSE:
-To attach the microbe onto the slide gently so that it can be stained and be visible as microbes are colorless
PROCEDURE:
- Obtain a well-washed slide and draw a circle on the bottom at about 75% along the slide; lable the slide as to the microbe name abbrev. or colony number
- Heat/cool your loop and capture a drop of water in the loop and place it on the circled area on your slide.
- Remove the dust from the sample cap and tube lip by passing it rapidly through the flame (DO NOT FLAME COTTON OR FOAM PLUGS).
- Holding the cap in the same hand as your loop (pinky finger) move the loop into the tube about ½ way. Gently touch the flat side of the loop the to agar and pull slightly. Lift and withdraw the loop – this should take no more than 5 seconds You should have a very small amount of bacteria (like snot) on your loop.
- Quickly flame the lip of the tube once more replace the cap and flame the cap. Put the tube in the rack or block.
- Mix the material on your loop with the water and spread as much as possible.
- Air dry
- Heat fix
NOTE:
-Check the "density" of your smear using The "e" Test. You want it just barely cloudy - NOT CLOUDY! If it is too cloudy, make two or three smears out of one by transferring material to two more slides using your clean loop.
--------------------------------------------------------------------------------------------------------------------
Title: Preparation of a smear from BROTH (Concentrating the microbes in the liquid)
PURPOSE:
-To attach the microbe onto the slide with agar for staining as microbes are colorless
PROCEDURE:
1. Prepare well-washed slide and draw a circle on the bottom at about 75% of the way along the slide. Label the slide as to the microbe or colony number. Mix your broth culture by holding it by the glass and banging it a few times against your palm
2. Use the “e” test to decide the number of coatings before the HEAT FIXING STAGE
3. Heat/Cool your loop.
4. Remove any dust on the lip of the tube through flame.
5. Dip the loop of into the well mixed broth and smear it on the slide within the circle in a circular pattern. Stay within the circle. Air Dry & REPEAT several times according to the “e” test
6. Heat fix.
NOTE: The "e" Test is an "Eyeball" test for determining the concentration of a solution. Take any mixed fluid + microbe. Put it in a test tube or on glass slide. Then put the text printed letter "e" next to the glass. If the fluid cloudiness prevents you from reading the letter "e" easily, then the concentration is high. For making smears from a broth, very cloudy=2.5 coats, very clear broth=7-10 coats.
------------------------------------------------------------------------------------------------------------
PURPOSE:
-To observe size, shape and morphology of a microbe. Sometime used to see contamination of a pure culture.
PROCEDURE:
- Obtain a slide with a prepared smear.
- Cover the smear with Methylene Blue for one minute.
- Wash the excess off the smear with water until the water runs off light blue. DO NOT OVERWASH
- Shake the excess water off and blot it dry.
- View the slide under 10X and then oil immersion.
NOTE:
-Methylene Blue stains living cells light blue while dead cells stain black. Methylene Blue is toxic to cells after 15 minutes.
=========================================================
Click here for all the info on THE Gram Stain PROCEDURE
Title: The Gram Stain
PURPOSE: to detect the thickness of the cell wall (murein or peptidoglycan layers) of bacterial cells
1. Prepare a good thin smear (see smears above)
2. Cover smear with Crystal Violet for 1 minute (put a LOT of stain)
3. Rinse with water, gently
4. Apply Gram’s iodine solution (a 1/2 a dropperful or so), shake it off and reapply again; let set for 1 minute
5. Shake off the Gram's iodine solution and add 1 to 3 drops of ETOH (Ethyl Alcohol, Gram's Alcohol, or Acetone Alcohol) for 1-2 seconds
6. Rinse with water, gently
7. Add safranin (don't be cheap - use a lot; make a "dome" of stain), shake it off and reapply the same amount and let it set for about 2 minutes
8. Rinse with water gently and blot dry
STAIN/SOLUTIONS ORDER: Crystal Violet (1min); Iodine (1 min); ETOH (1 second); Safranin (2 min)
CAUTIONS: Bacillus may show purple "hot-dog" shaped rods with pink cell pieces underneath... only count what is on top... majority rules... When using solutions use a LOT; never let a solution dry; when you wash aim above the circle and let the water run down over the circle, aim around the circle but NEVER AT THE CIRCLE. DO NOT OVERWASH, wash gently until the water running off is pastel pink NOT CLEAR!
===================================================================
Click here for all the info on THE Acid Fast Stain PROCEDURE
Title: The Acid Fast Stain (alt proc)
Title: The Acid Fast Stain (alt proc)
PURPOSE: to detect the presence of a lipid layer outside the cell wall of 2 Genera of bacteria (Mycobacteria & Nocardia)
PROCEDURE:
1. Prepare a good thin smear
2. Cover smear circle with the primary stain (make a "bubble" of stain) - carbolfuchsin
3. pass the slide underside back and to through the flame a few times waiting when out of the flame and then do this again until you see withdrawal of the edge of the stain bubble slightly - you will see some steam and the edge will indicate some evaporation of the Primary stain - DO NOT OVERHEAT or evaporate or cook until dry using the Primary stain as this stains everything HOT PINK and ruins stain! All HOT PINK is always wrong.
4. SHAKE off the Carbolfuchsin (this is USUALLY our Carbolfuchsin is rather thin and almost clear so DO NOT WASH - shake it off) then go to step 5
5. Add 1/4-1/2 dropper-full of acid/alcohol for 1-2 seconds and rinse immediately with water around the sides NOT at the stain
6. Add a "Dome" of methylene blue to the smear, shake it off and reapply a goodly amount and let it set for 3-4 minutes (if the stain turns purple instead of pink, shake it off and reapply the methylene blue)
7. Gently rinse and blot dry add immersion oil to the stained prep and view under 10x and then 100x
STAIN/SOLUTIONS ORDER: Carbolfuchsin (heat gently until edge withdraws) heat in a flame --------wash -> Acid alcohol 1 second à wash ->methylene blue 2 minutes
Carbolfuchsin (heat gently until edge withdraws) heat in a flame --------wash -> Acid alcohol 1 second à wash ->methylene blue 2 minutesCAUTION Notes: do not overheat! do not use acetone/alcohol; 1 hot pink tiny rod is positive, all hot pink and no baby blue rods means that you heated the Carbolfuchsin too much. We deciding look all over the whole slide because many times the tiny blue rods have one hot pink rod in another area... Bacillus may show blue "hot-dog" shaped rods with hot pink endospores... that is negative because endospores are NOT cells
===================================================================
Click here for all the info on the Endospore Stain (Alt. Method) Procedure
Title: The Endospore Stain (alt proc)
Title: The Endospore Stain (alt proc)
PURPOSE: to detect the presence of Ca++ ions present in the 2 Genera Bacillus (aerobic) & Clostridium(anaerobic) that produce endospore survival structures - THEY ARE NOT Reproductive!
PROCEDURE:
PROCEDURE:
1. Prepare a good thin smear
2. Cover smear circle with the primary stain (make a "bubble" of stain) - malachite green
3. pass the slide underside back and to through the flame several times waiting when out of the flame and then do this again until you see withdrawal of the edge of the stain bubble slightly and a little steam rises - DO NOT OVERHEAT or evaporate stain completely (ruins stain!)
4. Rinse gently with water to remove excess stain until the water runs barely green - NOT CLEAR!
5. Add the counter stain, saffranin, shake it off and reapply a good bit and let it set for 3-4 minutes (if the stain turns blue, then shake off and reapply the saffranin
7. Gently rinse with water (do not AIM at the smear) and blot dry
STAIN/SOLUTIONS ORDER: Malachite green heat in a flame --------wash à wash -> saffranin 2 min
CAUTION Notes: DO NOT OVERHEAT and dry out with primary stain; do not use any alcohol decolorizer; ANY "holes" in pink rods is positive; red and green tiny rods of the same size is negative, pink rods too small to have holes are negative, GREEN things of different sizes is negative stain debris and garbage, egg-shaped green things with larger pink rods is positive; ALL GREEN is usually wrong as it was overheated on the primary stain
===================================================================
Title: The NEGATIVE or Capsular STAIN(?) - Note: not really a stain because it doesn't color the microbes only the slide and the microbes remain clearish
Purpose: To detect whether bacteria have capsules made of "sugars" which do not take a stain well... the slide is colored while the light clear bacteria DO NOT Stain - thus this prep is really NOT A STAIN!
Procedure:
- 1 well-washed slide and another slide #2 can be dirty.
- Draw a small circle at 80% along the bottom of a clean slide
- Add 1 or 2 drop of Nigrosine dye.
- Mix plaque obtained with a toothpick from between your tooth and gum (DO NOT GET GUM TISSUE) with the nigrosine using a toothpick. Discard the toothpick in the regular trash – DO NOT LEAVE IT LYING ON THE TABLE; DO NOT DISCARD IN THE ORANGE BIOHAZARD TRASH!
- Moving from the short end of the slide, just touch the wet drop of Nigrosine with the second slide along its edge of #1 slide. The dye should spread across where the two slides touch. Lower the second slide and drag the #2 slide along the #1 slide. Apply some pressure but not too much, the smear should NOT be solid black as light will not penetrate the dye
- AIR DRY add a drop of immersion oil, then view it under 10X. Then oil immersion.
CAUTIONS/COMMENTS: Look for shadowy or lighter shapes of cocci and rods. Large bubbles are bubbles, jagged lines are cracks, the bacteria will be smaller than a broken pencil lead. Have your instructor sign your lab book if you are focused at 100x. Capsules do not stain as they are “sugar-like” compounds. This is not really a stain as it stains not the microbe but the slide. Capsules are invisible to the immune system and thus make a microbe more harmful. These bacteria cause tooth decay as they attach and the acid they produce from "eating" your food destroys tooth enamel.
HELPFUL VIDEOS and MY LIST OF STEPS:
VIDEO BOX NOT SHOWING UP BELOW?
VIDEO BOX NOT SHOWING UP BELOW?
Preparation of a Smear:
(BEFORE STAINING) A GOOD SMEAR (thickness!) is smear "B" on this photo
This little fan will save you 1/2 the time (SAFELY) in the Prep of a Smear... the second to last step is AIR DRY!
From AGAR (see the bottom of this page for a shorter card-type procedure):
- Wash your hands, disinfect your table-top and get into your incomplete Universal Precautions "outfit'
- OBTAIN a non-broken non-cracked slide. WASH the slide with detergent several times - until the water does NOT bead when it runs off the slide
- LABEL the slide with the name of the microbe to be stained
- Using your "SHARPE," DRAW a dime-sized circle in the center of the slide on the BOTTOM side of the slide
- HEAT your loop to red hot (the whole wire and 0.5 inches of the handle all red at once) and allow it to COOL for 15 seconds.
- OBTAIN a WATER bottle. Squeeze one drop of water and "capture" that drop in the cooled loop.
- PLACE the drop of water inside the drawn circle on top of the slide & spread it around inside the circle
- HEAT your loop to red hot (the whole wire and 0.5 inches of the handle all red at once) and allow it to COOL for 15 seconds.
- Quickly pass the top of the test tube slant through the flame to remove the dust and microbes from it (DO NOT FLAME FOAM or COTTON STOPPERS THEY CATCH FIRE!)
- REMOVE the top with your little finger (opposite hand from that holding the cooling loop)
- Quickly pass the LIP of the test tube through the flame to kill any microbes hanging to the lip of the tube
- ORIENT the tube with the slant at a FLAT angle and with the agar down inside the tube
- MOVE the cooled loop into the test tube going PAST the area where the microbe is growing on the slant (the microbial growth looks like "snot") and bring your loop down GENTLYonto the slant and PULL back scraping a very very small amount of microbial growth into your loop
- FLAME the lip of the tube after withdrawing the loop.
- FLAME the top of the test tube after replacing the top with your little finger & replace the test tube in a test tube holder - CAUTION: NEVER EVER LAY A LOOP ON YOUR COUNTER - IT MUST BE IN USE OR IN YOUR HAND! NEVER LAY A TEST TUBE DOWN ON YOUR COUNTER TOP - IT SHOULD BE IN YOUR HAND OR IN A TEST TUBE HOLDER! A TOP SHOULD BE IN YOUR HAND OR ON THE TUBE - NEVER ON A TABLE TOP
- MIX the material on the loop with the water on the slide. SPREAD this mix totally around the circle and BEYOND the circle... the more you SPREAD it the quicker it will DRY! Check the "density" of your smear using The "e" Test.... Remember... you want it just barely cloudy - NOT CLOUDY! If it is too cloudy, make two or three smears out of one by transferring material to two more slides using your clean loop...
- AIR DRY the slide - DO NOT HURRY THE AIR DRYING PROCESS! SPREAD the water + microbe mix out over the whole area beyond the circle - widely... the more area, the quicker your smear air dries...
- PICK-UP the air-dried slide in with your gloved fingers and pass the circled area DIRECTLY INTO THE BLUE AREA OF THE FLAME of your Bunsen Burner QUICKLY! This is called HEAT FIXING!
- TOUCH the bottom of the slide to your NAKED wrist. If it is warm, do not re-flame the slide as it would burn your skin to do it again and EXPLODE the bacteria.
===========================================================
From BROTH:
- Wash your hands, disinfect your table-top and get into your incomplete Universal Precautions "outfit'
- OBTAIN a non-broken non-cracked slide. WASH the slide with detergent several times - until the water does NOT bead when it runs off the slide
- LABEL the slide with the name of the microbe to be stained
- Using your "SHARPE," DRAW a dime-sized circle in the center of the slide on the BOTTOM side of the slide
- MIX your liquid broth culture of microbes by gently "banging it against your palm" 15X - USE THE "e" Test to determine how many coats of material you will need. Very cloudy = 2-5 coats or very clear broth = 7-10 coats...
- HEAT your loop to red hot (the whole wire and 0.5 inches of the handle all red at once) and allow it to COOL for 15 seconds.
- Quickly pass the top of the test tube slant through the flame to remove the dust and microbes from it (DO NOT FLAME FOAM or COTTON STOPPERS THEY CATCH FIRE!)
- REMOVE the top with your little finger (opposite hand from that holding the cooling loop)
- Quickly pass the LIP of the test tube through the flame to kill any microbes hanging to the lip of the tube
- Use your loop to obtain a 'loop-full' of liquid broth. PLACE the loop-full of broth inside the drawn circle on top of the slide and smear it around inside the circle... you can go outside until the liquid is dry
- AIR DRY the liquid on the slide COMPLETELY!
- Do this COATING with broth AND the AIR DRYING at least 7 times! NEVER HEAT FIX BETWEEN "COATINGS!"
- FLAME the lip of the tube after withdrawing the loop.
- FLAME the top of the test tube after replacing the top with your little finger & replace the test tube in a test tube holder - CAUTION: NEVER EVER LAY A LOOP OR A TOP ON YOUR COUNTER TOP - THE TOP SHOULD BE ON THE TEST TUBE OR IN YOUR HAND! NEVER LAY A TEST TUBE DOWN ON YOUR COUNTER TOP - IT SHOULD BE IN YOUR HAND OR IN A TEST TUBE HOLDER!
- AFTER THE 7TH COATING and the slide is completely AIR DRY, PICK-UP the air-dried slide in with your gloved fingers and pass the circled area DIRECTLY INTO THE BLUE AREA OF THE FLAME of your Bunsen Burner QUICKLY! This is called HEAT FIXING!
- TOUCH the bottom of the slide to your NAKED wrist. If it is warm, do not re-flame the slide until it would burn your skin to do it again.
YOUR SLIDE IS NOW READY FOR STAINING!
CAUTION! DON'T STAB THE AGAR WHEN PREPARING A SMEAR!!
---- put a card at the beginning of your Flash Cards that states that you will perform all the experiments aseptically and in the Incomplete or Partial Incomplete Precautions as well as performing the required handwashing and table-top disinfection before and after your work in lab. That you will not: lay down a loop or test tube but always have them in a stand or block and that you will always heat the cap, lip and loop (entire loop) when transferring bacteria... 1) Wash and rinse the slide well or until the water sheets-off the slide; place a small circle about the size of your little fingernail on the underside of each labeled slide at about 75% down the slide with a black sharpee 2) Heat and cool the entire loop, capture a drop of water in the loop 3) apply the drop to the top of the slide at the circle; afterward heat and cool the loop 4) transfer bacteria from an agar surface (DO NOT CHUNK THE AGAR) using a loop. Obtain the smallest amount of bacteria you can still see in the loop 5) using the loop, mix the bacteria in the water and spread it as widely as possible (it should be JUST barely cloudy when mixed) 6) Air Dry 7) Heat Fix then stain as desired ===MY GOOD CARDS EXAMPLES AND SIMPLE STAIN===
Preparation of a smear from AGAR (You are Diluting to get 1-100 microbes)
PURPOSE:
-To attach the microbe onto the slide with agar for staining as microbes are colorless
PROCEDURE:
NOTE:
-Check the "density" of your smear using The "e" Test. You want it just barely cloudy - NOT CLOUDY! If it is too cloudy, make two or three smears out of one by transferring material to two more slides using your clean loop.
--------------------------------------------------------------------------------------------------------------------
Preparation of a smear from BROTH (Concentrating the microbes in the liquid)
PURPOSE:
-To attach the microbe onto the slide with agar for staining as microbes are colorless
PROCEDURE:
1. Prepare well-washed slide and draw a circle on the bottom at about 75% of the way along the slide. Label the slide as to the microbe or colony number. Mix your broth culture by holding it by the glass and banging it a few times against your palm
2. Use the “e” test to decide the number of coatings before the HEAT FIXING STAGE
3. Heat/Cool your loop.
4. Remove any dust on the lip of the tube through flame.
5. Dip the loop of into the well mixed broth and smear it on the slide within the circle in a circular pattern. Stay within the circle. Air Dry & REPEAT several times according to the “e” test
6. Heat fix.
NOTE: The "e" Test is an "Eyeball" test for determining the concentration of a solution. Take any mixed fluid + microbe. Put it in a test tube or on glass slide. Then put the text printed letter "e" next to the glass. If the fluid cloudiness prevents you from reading the letter "e" easily, then the concentration is high. For making smears from a broth, very cloudy=2.5 coats, very clear broth=7-10 coats.
------------------------------------------------------------------------------------------------------------
The Simple Stain
PURPOSE:
-To observe size, shape and morphology of a microbe. Sometime used to see contamination of a pure culture.
PROCEDURE:
NOTE:
-Methylene Blue stains living cells light blue while dead cells stain black. Methylene Blue is toxic to cells after 15 minutes.
--------------------------------------------------
CARDS & PAGES YOU NEED FOR LP1 EXAM: Focusing the scope (Click here): NEGATIVE Procedure Stain? (Click here): SIMPLE STAIN (Click here): GRAM STAIN! (Click here): ACID FAST STAIN (Click here): ENDOSPORE STAIN (Click here): STAINING PROBLEMS (Click here): STAIN GAME (Click here): STAIN GAME ANSWERS (Click here): |
