IMPORTANT STAINING INTERPRETATION INFORMATION!
STAINING (BELOW) READ THIS COMMENT SECTION for your tests)...
COMMON FOCUSING ERRORS:
(1) Many of those that couldn't focus under 100X had nothing on their slides and were wasting time trying to focus under 100X on NOTHING - they didn't wash their slides - I CHECKED EVERY SLIDE and only 2 of 39 were washed well enough. A few people were not heat fixing hot enough. One was over heat fixing.
(2) Most people did not know WHERE their condenser ring was and did not LOOK under the stage to find it; most condensers were not up nor was the iris open
(3) Most students spent hours trying to focus on 100X but they didn't "spin" the fine adjustment many times one way and then the other - so they never "arrived" at the focal point. LISTEN! If you cannot focus on 100X in 5 min. START OVER AT 10X & do not EVER EVER use 40X or 100X to start! Don't spend more than 5 min trying to focus on 100X. Re-do the slide (WASH IT THIS TIME) or start focusing again at 10X. Never touch the Course Adjustment Knob when on 100X!
COMMON STAINING ERRORS:
(1) 95% were NOT washing their slides (and testing to see if their slides were really clean) - just going through the motions is NOT washing. Did you test the slides for beading/sheeting? If you don't wash it, nothing will stick and you will spend hours focusing nothing!
(2) On the ACID FAST STAIN - there is NO time for the Carbolfuchsin - DO NOT LET IT DRY OUT; DO NOT EVAPORATE it all... just pass it through the flame and wait after each pass - you are not to continuously pass the slide through the flame - it will get TOO HOT! Pass and wait until the 4 drops of primary stain "withdraw" a little - evaporate a little! Then use 1/4 - 1/2 dropper full of Acid Alcohol (not 2-4 drops) above your circle and wash immediately. Apply the Methylene Blue and shake it off; if you didn't get enough "red" off you may have to shake-off and reapply the Methylene Blue several times. If you get purple (red+blue=purple) it is wrong; all red is wrong too.
(3) MANY people read the oval egg-shaped HOT PINK endospores as Acid Fast +; ENDOSPORES are NOT CELLS! They do not count... Large baby blue rods with hot pink oval endospores is non-acid fast (AF-).
(1) Many of those that couldn't focus under 100X had nothing on their slides and were wasting time trying to focus under 100X on NOTHING - they didn't wash their slides - I CHECKED EVERY SLIDE and only 2 of 39 were washed well enough. A few people were not heat fixing hot enough. One was over heat fixing.
(2) Most people did not know WHERE their condenser ring was and did not LOOK under the stage to find it; most condensers were not up nor was the iris open
(3) Most students spent hours trying to focus on 100X but they didn't "spin" the fine adjustment many times one way and then the other - so they never "arrived" at the focal point. LISTEN! If you cannot focus on 100X in 5 min. START OVER AT 10X & do not EVER EVER use 40X or 100X to start! Don't spend more than 5 min trying to focus on 100X. Re-do the slide (WASH IT THIS TIME) or start focusing again at 10X. Never touch the Course Adjustment Knob when on 100X!
COMMON STAINING ERRORS:
(1) 95% were NOT washing their slides (and testing to see if their slides were really clean) - just going through the motions is NOT washing. Did you test the slides for beading/sheeting? If you don't wash it, nothing will stick and you will spend hours focusing nothing!
(2) On the ACID FAST STAIN - there is NO time for the Carbolfuchsin - DO NOT LET IT DRY OUT; DO NOT EVAPORATE it all... just pass it through the flame and wait after each pass - you are not to continuously pass the slide through the flame - it will get TOO HOT! Pass and wait until the 4 drops of primary stain "withdraw" a little - evaporate a little! Then use 1/4 - 1/2 dropper full of Acid Alcohol (not 2-4 drops) above your circle and wash immediately. Apply the Methylene Blue and shake it off; if you didn't get enough "red" off you may have to shake-off and reapply the Methylene Blue several times. If you get purple (red+blue=purple) it is wrong; all red is wrong too.
(3) MANY people read the oval egg-shaped HOT PINK endospores as Acid Fast +; ENDOSPORES are NOT CELLS! They do not count... Large baby blue rods with hot pink oval endospores is non-acid fast (AF-).
HINTS FOR EXAM QUESTIONS:
If you see a "river" under the scope = did not HEAT fix enough in prep of the smear
If you see a "tire tread" appearance or the microbes "fat" mishapen and look melted and fused together = over heat fixed the smear
If you see SOMETHING at 10X but NOTHING at 100X = Overwashed the slide, under heat fixed the smear, no immersion oil or too much or you need to clean your 100x objective or you "flipped" the slide onto the wrong side for focusing!
If you see something at 10X and the view at 100X is not clear and crisp = (1) not enough oil (add 1 drop the max amount should be 2 drops on the slide - DON'T FRY BACON!) or (2) too much oil (raise the objective and clean it with lens paper and re-focus under 10X and then move to 100X and fine focus again) or (3) your slide is slightly over washed - do it again or close the iris diaphragm slightly
GREATEST SIN in Microbiology = "CHUNKING" the agar (the bacteria is the smooth "snotty" like material grown on top of the gel agar. You should move past the area you wish to sample with your heated cooled loop and "brush" the agar surface very very lightly. You should be barely able to tell you have ANYTHING on your loop... mix it with 1 drop of water on a CLEAN slide to barely cloud as air drying, then heat fix to very warm and stain
If you see bubbles at 10X and 100X = you didn't blot the slide dry with paper towel or bilbulous paper
If you lots of various sized shapes and very small drops of dye = you stained on the dirty rack, blotted with dirty paper, or didn't wash well
THE COLORS ARE WRONG! If your scope light is low and yellow then you cannot determine the TRUE color of the stain or see "holes" and your evaluation will be wrong!!! USE THE LIGHT FULL ON AT EVALUATION! THE CONDENSER UP!
The most common error in focusing a slide under 100X is failing to raise the condenser to the back of the slide
The important step in making a SMEAR (and stain) is WASHING THE DANG SLIDE UNTIL THE WATER "SHEETS OFF" NOT beads-up ------ TEST EVERY SLIDE under a faucet!!!!!
IF EVERYTHING IS ONE BRIGHT COLOR = always WRONG!!! (using a primary stain color) and you are heating the slide = YOU ARE HEATING TOO MUCH! If when you make the AF stain you see NO TINY BLUE RODS but all HOT PINK TINY RODS you are over-cooking on the primary stain (just a little evap needed)
If you get PURPLE on the Acid Fast Stain or BLUE on the Endospore Stain = you are not washing enough of the primary stain off or you overheated the primary stain or you didn't use a 1/4 dropper-full of acid alcohol on the AF Stain and then wash well
If you see black or dark grey shapes = you put your Sharpee mark on the top of the slide and not on the bottom and your look "scraped" it off
IF it is too dark on viewing = raise the condenser and open the iris or turn up the rheostat power wheel
========================STUDY SHEET FOR Lp1======================
Date: March 13, 2012 2:59:36 AM PDT
Subject: Microscopes - pros/cons
Microscope types:
1. Compound bright/light field & darkfield microscopes
2. Fluorescence microscope
3. Phase contrast microscope
4. (Differential) Interference aka Nomarski microscope
5. Scanning electron microscope
6. Transmission electron microscope
Under each heading, I marked which microscopes apply by the corresponding number as listed above.
View Motility:
1, 3, & 4 (note: the compound light microscope (#1), can be used to
view motility by making it into a "darkfield" microscope by lowing the light power and closing the iris.
Therefore, those that cannot be used to view motility are 2, 5 & 6. Fluorescence scopes use dyes that kill the microbes as do the electron microscopes.
2-D:
1 (light & darkfield), 2
3-D:
3, 4, 5 & 6
Shows chemistry (color):1 (compound LIGHT FIELD ONLY)
Shows NO chemistry:
1 (compound DARKFIELD ONLY), 2, 3, 4, 5 & 6
Shows internal structure:
1 (compound BRIGHT FIELD ONLY), 4
Show NO internal structure:
1 (compound DARKFIELD ONLY), 2, 3, 5 & 6
The difference between Practice Lab Practical 1 and Lab Practical 1 is this: ON PLP1 there are only 50 Multiple Choice questions on Ch3, Smears, Stains, and Safety... and it counts only 200 points of weekly test 1000. Of the 1000 Weekly points in the class the average only counts 25% of your final grade in the class and I drop the two lowest 100 pts tests. Also, you can discover your errors on PLP1 and "fix" them in the final stain practice sessions in Micro-40 this coming Friday 9-1. The REAL LAB PRACTICAL 1 EXAM has 100 multiple choice questions and counts 20% of your Final Class grade alone... it cannot be dropped.
Call the Prof is every Sunday by GOOGLE+ from 12-2 PM for Q and A! is by telephone every Sunday from noon to 3 PM. Email is anytime.
Lab Practical 1 is: LP1 is the first major Microbiology Exam. LP1 is valued at 20% of your Final Class Grade. LP1 covers: making (from memory) good smears (make 10 in 15 min) and 3 good stains for the 3 major stains in Micro (Gram, Acid Fast, and Endospore). Lab Safety, reading and interpreting stains (photos) and anything from the notes, videos and lectures of Chapter 3 - The Microscope.
LP1 has 4 parts:
(1) 100 multiple choice questions from Ch 3, smears, stains and safety
(2) making 10 good smears from 1 unknown slant in 15 min without agar chunking (I will check each tube)
(3) making, focusing and reading good examples of the 3 major stains (NOT TOO THICK OR THIN) and
(4) being able to interpret hotos of difficult or problem organisms on each of the 3 stains... pos/neg and rod/cocci (see Problem Staining and The Stain Game online NAV page).
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NO CARDS or NO NOTES in LABS after the first week of stains!!!! Everything must be memorized! If you want the PowerPoints used in class to discuss the 3 major stains - here they are:
IMPORTANT VIDEOS: check it out and join my Google+ circle (Students)
A good smear always precedes STAINING:STAINING!This is a good smear (barely cloudy before and AFTER drying and heat fixing) see smear "B" in this photo: note you can use the "e" test to determine proper concentration in water from agar or a broth although it is most often used to determine the number of coatings for a broth smear....
NOTE: the dyes we use are PERMANENT! Do NOT spill them or "slop" them on the table-tops or floor. If you spill a dye, obtain the RED Acid Alcohol Bottle on the Discard Cart and remove the dye with this and a paper towel!!!
Microbes are not only very very small they are actually COLORLESS! Thus, for us to visualize them under a brightfield light microscope using 1000X oil immersion, we must give them "CONTRAST" or color the background instead of the microbe as in the Negative or Capsule staining.
The process of adding contrast to a microbe also involves ATTACHING the microbe to the slide as well as 'coloring' it... THIS attachment process is known as Preparation of a Smear.
Smear preparation is generally accomplished in one of two ways according to the media on which or in which the microbe is presented :
(1) with the microbe growing in a test tube on top of a mass of 'slanted' agar media then it is "FROM AGAR" and
(2) if the microbe is presented growing inside a liquid it is "FROM BROTH"
CAUTION!!! Microbes grow in a film on TOP of the agar - don't stab the agar with the loop!!!!
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| CAUTION! When preparing a SMEAR - DON'T TRY AND STAIN THE AGAR! |
REMEMBER:
- All staining is done AFTER SMEAR PREPARATION!
- The colored stain used usually involves several colored "dyes" with specific chemical properties that relate to the microbes structure and thus help identify the microbe being stained!
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READ THIS file: You get one "pass on the scopes" ---- after that you are penalized 10 PQ points per event and when you reach "0" I will begin subtracting points from you weekly score. DO NOT LET THIS HAPPEN AGAIN!
