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
- 1/2 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-).
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
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
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======================
From: Christina Fieber <email@example.com>
Date: March 13, 2012 2:59:36 AM PDT
To: "Mexistentialism@yahoo.com" <Mexistentialism@yahoo.com>
Subject: Microscopes - pros/cons
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.
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
1 (light & darkfield), 2
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 12-3 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.
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).
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:
check it out
and join my Google+ circle (Students)
A good smear always precedes 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....
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
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.
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
(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!!!!
|CAUTION! When preparing a SMEAR - DON'T TRY AND
STAIN THE AGAR!
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!
In this course our staining/smear
procedures also kill the
microbe while at the same time attaching the microbe to the slide and adding
enough contrast for us to visualize the microbe.
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