STAINING (BELOW) READ THIS COMMENT SECTION for your tests)...
Video on how to focus the scope:
Focusing a scope under 100X Oil
Focusing the Scope (click here)
HOW TO FOCUS THE MICROSOPRE (STEP BY STEP):
(1) obtain your seat #/assigned scope from the cabinet; carrying it with both hands
(2) check the power switch (and rheostat light level switch if applicable) and make sure bot are off or at the lowest setting
(3) plug in the scope and move the light setting to 100% and then back a little to about 90%
(4) place your slide on the stage inside the cut-out slide holder. Use the stage controls to move your slide directly over the hole in the stage. Put your "circle" or colored area of the slide directly over the hole in the stage over the condenser light. Make sure you have already added immersion oil to the slide circle/colored area.
(5) check for the correct condenser settings: use the condenser height adjustment knob (usually a black know found on the right side of the scope just below the stage) so that the condenser apparatus is moved up to almost touching the back of the slide. Make sure that the iris diaphragm lever or ring open to 90%
(6) move the eye width settings on the binocular scope to their widest and put your eyes 1-1.5 inches (2.5 to 3 cm) away from the eyepiece opening. Bring the two oculars toward or closed until the 2 white circle just join to form 1 white circular backgound
(7) using the objective nose-piece RING, click the 10X LOW POWER objective into place (CLICK IT!)
(8) using the large COURSE ADJUSTMENT knobs on the base of the scope to bring the stage all the way up - stop when there is resistance from the knob - DO NOT FORCE the COURSE ADJUSTMENT KNOB when all the way down or up! REMEMBER Biol 112 people you must UNLOCK the COURSE ADJUSTMENT lever - it is printed on the level
(9) Place your eyes near the oculars/eyepieces (about 1.5 inches or 3 cm BACK). Turn the COURSE ADJUSTMENT KNOB DOWN slowly - it may take from 3-7 full slow turns - until you see a flash of color or clearing of the field. NOTHING LARGE OR CLEAR BUBBLE LIKE is correct! LARGE items are dust and garbage --- so, move up or down and away from them to see the real bacteria which are very very small (about the size of a broken pencil lead at 1000X!). If you have trouble, try to focus on the SHARPEE mark which should have been placed on the bottom of your slide during smear preparation. IF YOUR SMEAR IS TOO THICK OR YOU HAVE AGAR ON THE SMEAR YOU WILL ONLY SEE MASSES OF DARK COLOR - WRONG!
(10) once you see the "flash" of color or clearing - use the FINE FOCUS or SMALLER inside knob inside the course adjustment know to bring the focus into perfect clarity. If you see something that you are especially interested in viewing at 1000X, use the stage adjustment knobs to put it just off the end of the pointer in your ocular viewer(11) using the NOSEPIECE ring, move your objective lens COUNTERCLOCKWISE 2 spots or clicks (so that you do not drag the 40X objective into the immersion oil) so that the oil immersion 100X objective slides into the drop of oil. DO NOT PANIC, it will NOT break the slide if you have really focused well at 10X! Then, USING ONLY THE SMALLER FINE FOCUS KNOB, finish focusing your scope to clarity at 1000X. NEVER NEVER EVER USE THE COURSE ADJUSTMENT KNOB WHEN YOU HAVE THE OIL IMMERSION LENS CLICKED INTO PLACE - start over at 10X
OTHER SCOPE FOCUSING... ADJUSTMENTS/COMMENTS/CONSIDERATIONS:
(a) At 100X, you may need to move the stage adjustment knobs some to search the slide (AF stains may have only 1 positive rod and if you do not look over your entire slide you might miss the question).
(b) You may also need to bring your light to max and white to determine true cell staining color or
(c) you may need to reduce the Iris diaphragm to improve your resolutions some and prevent FLARE.
(d) be careful you are not looking at SHARPEE scraped off marks if you put the Sharpee circle on the wrong side when making your smear
(e) if it just won't focus under 100X first see if you are on the correct side of the slide (smear side up), next re-start under 10X and try again, next lower the stage and clean the oil immersion lens with lens paper as they often get "gooey" and finally check that your condenser is all the way UP!
IF NOTHING WORKS CALL THE INSTRUCTOR THE SCOPE MAY BE DAMAGED!
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) Many 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 extra 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. Did you "search your whole circle area or just focus at one spot?"
(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-).
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 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, 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
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! Don't be a baby!
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" & DOES NOT bead-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======================
Subject: Microscopes - pros/cons
Microscope types:
1. Compound bright/light field & darkfield microscopes
2. Fluorescence UV microscope
3. Phase contrast refracting 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
DME SCOPE FAULTS:
Be aware that every microscope has a weakness. On our DME scopes the location, the operation and proper adjustment of the IRIS DIAPHRAGM RING is a liability as students often do not look under the stage and often "GRAB" the condenser apparatus instead of the Iris RING to increase the illumination - doing this results in a condenser, filter and condenser lens misalignment plus the condenser apparatus will come loose & even fall out of the scope rendering your scope useless. Repairs take several hours and are difficult. Please LOOK beneath the stage and then adjust your Iris diaphragm ring if your field of view is dark or fuzzy.
PLEASE DO NOT GRAB THE CONDENSER. USE THE RING TO OPEN and CLOSE the light path (to obtain more or less light) also use the lower right small black knob (beneath the stage) to raise and lower the condenser. Raise the condenser apparatus for lab work and lower it 1 turn for storage.
OLYMPUS SCOPE FAULTS:
There is a LOCK on the right side of the Course Adjustment Knob of the
scope... if the lock lever is left at 90 degrees (down) instead of "un-locked"
or vertical... moving the course adjustment knob will STRIP the gears of the
scope. From that time on the stage will drift DOWN each time you fine
focus... making observation difficult.
PROPER STORAGE OF THE SCOPE:
1) Turn off the light by using the bight red power wheel located on the lower right side of the scope foot. Pull the red wheel toward you until it clicks for OFF; the hot red light becomes dark inside the lighted wheel.
2) Lower the stage completely and remove the oil from the 2 highest objectives
(Olympus stage must be left UP). Both the 100X OIL Immersion lens and the 40X objective lens must be cleaned with LENS PAPER ONLY! You may use ethanol on a Q-tip to remove extra oil. Use the nosepiece RING to click the shortest 4X (shortest red lined objective) into place for storage.
3) Lower the Condenser Apparatus 1 turn using the small black knob just below the stage on the right-hand side of the scope.
4) Move the slide adjustment "arms" on the stage so that they do not extend beyond the stage.
5) REMOVE any "splatters of oil or stain from the stage, arm and foot of the scope with a paper towel moistened with Acid Alcohol.
DO NOT USE PAPER TOWEL ON ANY GLASS PART OF THE SCOPE.
6) Wrap the cord using the cord holder and take it to your AFTER CLASS SCOPE CHECKER...
7) Have your AFTER CLASS SCOPE CHECKER (these checkers are assigned by all instructors) confirm your proper care of the scope before you put the scope in the correct numbered spot.
The checkers look over your work & check that the oil is removed from the 40X and 100X objectives, they do not DO IT. You do!
HINTS AND COMMENTS ON THE PRACTICE LP1 and LP1:
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+ HANGOUT from 12-3 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.