Download Lab 1 Instructions

LAB SAFETY: Open-toed shoes and shorts above the knee are not allowed since a spill can
cause skin irritation. Put your backpack, purse, etc in the racks at the back of the room.
Before leaving, make sure your area has been disinfected with desktop cleaner, left clean,
push your chairs in, and that the microscope has been put away properly. If it is found not
properly put away, you will receive a note indicating what was wrong with it.
The lab counters must all be wiped down with disinfectant when you come in and when you
leave. You may throw the paper towels into the regular trash afterwards.
Get a white tray from the back of the room and always put your plates and racks of tubes on
the tray so they are not directly on your desk top. When staining a slide, get a grey bucket
from the back of the room and place it on the white tray. Then get a 250 ml beaker, place it in
the grey bucket. Place your slide on top of the beaker so the beaker catches most of the stain
that drips off it. The grey bucket will catch the rest of the stain. The stain waste is then
dumped into the white containers on top of each of the two lab rows.
Dispose of cover-slips and glass slides in the red sharps container. Most fires in this lab are
started from alcohol becoming ignited. Just get away from it, cover it, and let it burn out.
Remember, a lab coat can be taken off and used to put out a fire if someone’s hair catches
fire, etc.
There is no food, drink, gum or water bottles allowed in a micro lab.
The biohazard bag is for any and all hazardous materials, including a toothpick you put in
your mouth, etc. Use the regular trash for everything else.
For the glassware flasks and tubes put them on the indicated tray on the left side of the room.
If there is a bacterial spill, cover it with paper towels, saturate them with disinfectant
(Clorox) for ten minutes, then put those paper towels in the biohazard bucket.
First aid kit is in the cabinet near Lab Tech door.
Recent case of Salmonella infection that came from a teaching lab:
http://www.cdc.gov/salmonella/typhimurium-laboratory/011712/index.html
INSTRUCTIONS FOR TODAY
Download all of the instructions for the labs this semester from my website, and save them in a
folder. Make sure you bring ALL the semester’s instructions with you each week, because
we will use the instructions from previous weeks throughout the semester.
Divide into groups of 3-4. Each GROUP will get one nutrient broth tube, three nutrient agar
plates (Petri dishes), and 4 packs of sterile swabs (2 per pack). One person will be the group
leader who will put their name on the plates and tube.
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HOW TO INOCULATE THE BROTH
Label the tube with your group leader’s name and source of the swab. To inoculate the tube of
nutrient broth, moisten a cotton swab with water, swab the inanimate surface (fomite) you
want, then stick the whole swab in the tube and swish it around. Press the swab against the inside
of the glass to squeeze off the excess liquid, and discard the swab in the regular trash. Twist the
cap closed, then open it by ½ turn so air can get in. Place it in the rack with the tubes from the
rest of the class, and the whole rack of tubes will go in the incubator.
Take three NA Petri dishes per lab group. The lid is the larger (wider) side and the bottom is
the smaller side. Use the Sharpie or wax pen to write your group leader name on the bottom of
the Petri dish, around the edge in a ring.
On one plate, turn it upside down and draw a giant plus sign on the bottom so the plate is divided
into four quadrants. You will get a sterile cotton swab, add a drop of water to it, and swab a
surface to collect bacteria, one swab for each of the three plates and the tube. The plate with the
quadrant should be inoculated with a sample from your body (either skin, throat, hair, etc). The
other plates should be a sample from the environment (light switch, computer keyboard,
bathroom, water fountain, door knob, etc). Make sure you write your source on each plate and
tube. Instructions on how to inoculate the plates are on the next page.
The plates will stay out at room temperature in the Yellow Petri Dish rack except the sample
from your body will go in the Red Petri Dish rack, which goes in the incubator. The nutrient
broths will go into the incubator in one rack. Next time, we will examine the colonies and
practice using the proper terminology to describe the morphology (appearance) in your tube and
plates.
PLATE INOCULATION TECHNIQUES
To inoculate the plate, there are four different techniques, depending on what tests you will
perform on the subculture. Today, we will do 2 “streak for confluence” plates (on the fomite
swabs) and one “streak for isolation” plate on your body swab.
1) Streak for confluence Plate (use this technique today on the 2 fomite swabs)
2) Streak for Isolation Plate (use this technique today on your body swab on the plate with
quadrants).
3) Spread Plate (soil sample after dinner)
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a. A small amount (several drops) of a previously diluted sample is spread over the
surface of a solid medium (Petri dish) using a spreading rod, which is a sterile
glass or plastic rod bent at 90°.
4) Pour Plate
a. A small amount of diluted sample is mixed with melted agar and poured into
empty, sterile Petri dishes. A serial dilution of the bacterial sample is performed
first, then a small amount of each dilution is pipetted into the empty Petri dishes,
then the melted agar is added. This allows bacteria to be inoculate throughout the
media.
The spread plate and pour plate techniques are quantitative methods used to determine the
number of bacteria in a sample. We will use those techniques later in the course.
HOW TO PERFORM A STREAK PLATE FOR CONFLUENCE INOCULATION
Moisten a cotton swab with water and obtain one different fomite sample (inanimate object)
for each of the two nutrient agar streak plates. For example, swab your phone and inoculate
one plate and then another person uses a new swab on a doorknob and inoculate the other plate.
The inoculated swab is used to streak the sample many times in a zig-zag over the surface of the
agar in the Petri dish. It is streaked from top to bottom. The streaks should be fairly close
together.


Never leave a culture dish open, even for a short time when viewing colonies of
organisms. During your procedure, keep the lid close to the dish, open it only as far and
as long as is necessary to accomplish the procedure, and keep the lid between your face
(and your germs!) and the agar surface. Open it sideways, not so the opening is in front of
you where you can breathe on it.
Never place a Petri dish lid on another surface. You must hold the lid in your hand
during the transfer. Make sure the open side of the lid is facing downward and covers
the agar, so no contaminants can float down into it.
Streak for
confluence
This plate is WRONG
It should be a tighter
zig-zag, leave no agar
untouched. Then rotate
the plate and zig-zag
again to be sure.
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HOW TO PERFORM A STREAK FOR ISOLATION INOCULATION
(Do this on your plate with the quadrant drawn in)
Purpose of this technique: When a doctor takes a swab from an infected wound and sends it to
the lab, he needs to know what organisms are in the wound so he can select the proper antibiotic.
There usually will be more than one organism present, so the lab will need to take the mixture of
bacteria and separate them out into pure cultures before they can identify each organism.
The body swab will be used for the streak for isolation plate. Use the Petri dish with the four
quadrants drawn on the bottom (label each quadrant 1, 2, 3, 4 clockwise; put the numbers in the
bottom corner of each quadrant so it does not obstruct your view of the organisms next week). Obtain
your inoculum on the moistened cotton swab (unless you are swabbing mouth or throat, the swab can
be dry) and zig-zag it over the upper left quadrant (1) of the plate. Make sure your zig-zag strokes
are close together to get the most bacteria onto the first quadrant. Throw the swab out. Turn the
plate ¼ turn counterclockwise so the second quadrant is at the top. Get a new swab (no need to
moisten it; keep it dry) and drag it once across the middle of the first quadrant and into the top of the
second quadrant, then (without lifting it) zig-zag throughout the second quadrant, keeping the zigzags close together. Throw the swab out. Turn the plate counterclockwise so quadrant 3 is at top. Get
a new dry swab and drag it in one line from the middle of the second quadrant and drag it into the
third quadrant, and zig-zag that area. Throw that swab out. Get a new dry swab, turn the plate
counterclockwise, and drag the last line from the middle of the third quadrant into the fourth quadrant
and zig-zag.
When you use this technique, you are dragging fewer and fewer organisms into each quadrant, so
that the last quadrant will have individual colonies that do not overlap each other. If you were
successful, in the next lab period you will see separate colonies in the last quadrant. If you were
not successful, different looking colonies will still be overlapped in the last quadrant. What
might have gone wrong? Perhaps you had too many organisms on the first quadrant, or maybe
you dragged too long of a line from one quadrant to the next, or perhaps your zig-zags were too
narrow and did not cover enough surface area.
The next step (next lab period) would be to select the very best looking individual colony which
is separated from the other colonies, touch it with a sterile needle, and zig-zag it across the
surface of a slant. Do the same for each of the other colonies that look different from each other,
so you end up with one slant for each organism. You would then let those grow and run some
tests on them to determine what organisms are present.
Notice the zigzags are close
together
Streak for isolation
Step 2
Step 1
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Step 4
Step 3
INCUBATION
Always incubate plates upside down, so the agar is on the top. Why do we do this? It
prevents condensation from developing on the inside lid, and then dropping onto your agar,
mixing the organisms. It also enables you to read the important writing on the bottom of the
plate. We will incubate body swabs at 37° C, which is body temperature. The others will be
room temperature.
HANDWASHING WITH SALIVARIUS AGAR
Use a Sharpie pen to divide a Mitis Salivarius plate into three sectors and
mark them a, b, and c on the bottom of the plate. This agar has nutrients that
your saliva bacteria prefer.
1. Open the lid of the plate at 45 degree angle like a hinge and roll one
or two fingers across the agar in the “a” sector, like you are being
fingerprinted. Don’t rip the agar though. Lower the lid back in place.
2. Swab your mouth with a cotton applicator stick and rub the saliva
onto the same fingers and roll them across the “b” sector, like you
have licked your fingers and are now being fingerprinted.
3. Wash your hands with soap and water, dry with paper towel and roll
your same fingers across the “c” sector.
Incubate the plate at 37 degrees C.
Streptococcus salivarius is a species of Gram-positive cocci bacteria which colonize the mouth
and upper respiratory tract of humans a few hours after birth, making further exposure to the
bacteria harmless. These bacteria are opportunistic pathogens, causing dental caries and
endocarditis in the immunocompromised. Mitis salviarius agar is used to differentiate among
species of Streptococcus that are flora in the mouth. The sugars in this medium are sucrose and
glucose. The medium also contains the dyes trypan blue and crystal violet, which inhibit other
Gram positive and Gram negative bacteria. S. salivarius is able to use the sucrose in the media to
produce a capsule around itself. Strep salivarius uses the sugars to produce a gummy-like
capsule, producing sticky, mucoid, gum-drop colonies. Strep mitis colonies will be small, flat,
light blue. Strep mutans will be undule-shaped colonies, with a granular frosted-glass appearance
(making dextran from sugar). Enterococcus will produce dark, blue-black colonies.
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SOIL PROJECT
1) Don’t use really fertile soil…dry is better, but not in an area where no plants are growing. It is best to get
soil close to roots of nearby plants, and dig down about 2 inches first.
2) Fill a 15 ml test tube all the way up with soil.
3) Record the place of collection, temperature, and any of the below data you can collect
LOCATION
Location
Latitude
Longitude
Date and Time Collected *
Date
Time
Sample Site Descriptors *
SOIL SAMPLE
Air Temperature (°C)
Humidity (%)
Depth (In.)
Type of Soil
Soil Temperature (°C)
pH of Soil
Water Content (%)
4) Pour the whole 15 ml tube of soil into a 250 ml Erlenmeyer flask and fill it to the 100 ml
mark with de-ionized water in the large plastic jar by the sink. That gives a 15% soil to
water ratio. Stir it up to make it muddy.
5) EACH STUDENT will obtain one of each of the following two plates PER SOIL
SAMPLE: LBA (Luria-Bertani agar), and TSA (tryptic soy agar)
6) Label the BOTTOM side of the Petri dishes, around the EDGE, with your name, “soil”,
date, and instructor name.
7) Use a pipette to drop 0.5 ml of the solution (half a stem full of the pipette) onto a Petri
dish by opening the lid of the Petri dish at a 45° angle, keeping the lid over the agar.
Squirt the solution onto the top of the agar.
8) Use a sterile spreader, but you can use the same spreader for each of the three plates from
the same soil sample. Spread across the whole plate surface while rotating the plate to be
sure the whole surface is covered.
9) Make sure your plates all face upwards when you wrap your three plates together with
parafilm (don’t wrap each plate individually) and add a piece of tape on top with your
name on the tape. Then turn them upside down so the agar is on top. Leave these at room
temperature (yellow Petri dish holder). Do NOT put these in the incubator. The Tech
will put them in the refrigerator on Monday so they do not overgrow.
10) Clean up: throw out muddy water into the regular trash can. Put the muddy flask on the
tray to the left of the sharps container. Put your blue top plastic tubes that your soil was
in. Rinse the plastic spreader and put in on the tray where the flasks are.
HOW TO REMOVE YOU GLOVES
 Grasp the palm exterior of one glove with
your other gloved hand.
 Carefully pull the glove off your hand,
turning it inside-out.
 Hold onto that glove in your other, gloved
hand.
 Slide your ungloved finger into the opening
of the other glove at the wrist.
 Carefully pull the glove off your hand,
turning it inside out again. Now you can
grab it with your clean hand because you
are touching the inside of the glove.
 Discard in the trash can.
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HAND WASHING WITH GLOGERM™
1. Shake the bottle well.
2. Have your lab partner apply 5-6 drops of the gel on the palms of one of your hands.
3. Rub your hands together so the lotion covers all surfaces, including backs, between the fingers, and
up to the wrists. Scratch the palms with your fingernails.
4. Have your lab partner shine your hands with an UV-lamp to see the extent of coverage. Do NOT
shine U.V. light into anyone’s eyes!
5. Ask your partner to turn on warm water at the sink for you. Wash your hands with warm water and
soap the way you usually do.
6. Have your lab partner shine your hands with an UV-lamp to see the extent of coverage. Ask your
partner to record your observation in the table.
7. Repeat your hand washing in a thorough way, scrubbing your hands for at least 20 seconds.
8. Have your lab partner shine your hands with an UV-lamp to see the extent of coverage.
9. Repeat the procedure with your lab partner but with the roles reversed.
HOW TO WASH YOUR HANDS PROPERLY
It takes at least fifteen seconds to wash your hands properly – this is about how long it takes to
sing ‘Happy Birthday to You’ twice through!
Before you wash your hands, take a paper towel and put it under your arm.
Wet hands with water and leave the water running
Apply enough soap to cover all hand surfaces
Rub hands palm to palm, rub between fingers, and backs of hands, fingers, and wrists.
Rinse hands with water and do not turn the water off while drying your hands.
Dry thoroughly with the towel from under your arm.
Use the same towel to turn off the water faucet. Use the towel to open the bathroom door, and
then discard the towel in trash and exit without touching anything else.
LAB LECTURE MATERIAL
GROWTH MEDIUMS
Nutrients are added to agar (a seaweed product) and heated to a liquid, and sterilized. The sterile
liquid agar can be poured into a Petri dish and cooled. The Petri dish is then referred to as a
“plate.” Sometimes liquid agar is placed in a tube which is tilted 45° while cooling. This is called
a slant. Sometimes the nutrient medium is a broth which does not solidify. Tubes with this
medium are called nutrient broth tubes. All of these types of mediums are made for the purpose
of being inoculated. Intentionally introducing microbes onto nutrient agar and into nutrient broth
is known as INOCULATION, which means having a tiny amount of a bacterial culture streaked
across the surface. After inoculation, the plate or tube is usually incubated for at least 24 hours to
encourage growth of the sample. After 2-3 days, there will be too much growth, so we take it out
of the incubator and put it in the refrigerator to slow the growth.
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TYPES OF MEDIA
Culture Media Classification
1. By Consistency (broth, plate/slant, or gel)
2. By Contents
a. All-purpose (Nutrient agar = NA)
b. Isolation
i. Selective (selects for only certain types of organisms)
ii. Enriched (has extra nutrients (blood) for hard to grow species)
iii. Differential (allows to tell what type of organism is there; changes color)
Consistency refers to a liquid, solid, or semi-solid. The use of a semi-solid allows us to do two
things: see if the microbe is motile (can move), and to see if the microbe is aerobic or anaerobic.
ALL PURPOSE MEDIA
Nutrient agar (NA) does not support fastidious (hard to grow) organisms, so NA is a good allpurpose media. It will support the growth of a wide variety of organisms. It is also inexpensive.
ISOLATION MEDIA
There are three types of media used to isolate particular organisms
1. Selective media
2. Enrichment media
3. Differential media
Selective media
This type of media selects for a particular type of organism to grow. Selective media contain
chemicals that prevent the growth of unwanted bacteria without inhibiting the growth of the
desired organism. An example is Sabouraud’s Dextrose Agar (SDA), which has a high sugar
content and an acidic pH. SDA is called an ISOLATION MEDIA since it isolates molds and
yeasts, which do well in high sugar content, whereas bacteria are inhibited. Therefore, sugar acts
as a bacterial preservative; that’s why jams, jellies, and preserves don’t get bacteria growth.
However, molds are aerobic and they like sugar. They can get into your jelly jar when their
microscopic air-borne spores drift in whenever the jelly jar is opened. Toss out the whole jar!
Molds and yeast also grow well in an acidic environment.
SDA was originally designed to isolate molds of skin, nails, and hair, called dermatophytes,
which are opportunistic pathogens (they only cause disease if the skin is broken or the person’s
immune system is poor). Dermatophytes produce ringworm, especially athlete’s foot. Yeasts are
known for causing infections in women, diabetic and cancer patients. Since people with diabetes
have a high sugar content, they are susceptible to such infections. Scrape off a little skin and
place in SDA to isolate the microbe. SDA has 40 grams of glucose and a pH of 5.6.
Differential Media
Differential media contains various nutrients that allow the investigator to distinguish one
bacterium from another by how they metabolize or change the media with a waste product. Some
of these media contain a substance that turns yellow if one group of organisms are present, or red
if another group of organisms are present. Therefore, we can tell which group of bacteria are
there by the color the plate turns.
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Enriched Media
This type of media has added nutrients such as vitamins and amino acids. It is used to grow
bacteria which are hard to grow, known as FASTIDIOUS organisms. Many pathogens are
fastidious. An example of enriched media is Blood Agar, which has Trypic Soy Agar (made
from soy) and 5% sheep’s blood (defibrinated to keep it from clotting).
Broths and Slants
Broths are the same nutrients as NA, but no agar. They do not have to be boiled, just stirred.
They are placed in tubes instead of flasks and autoclaved as usual.
Slants are made exactly as NA, except they are poured into tubes instead of plates. After they are
removed from the autoclave, they are placed on slant racks to cool so the agar in the tube stays at
a slant.
Why use a slant?
We can inoculate just the top of a slant to get growth of aerobic bacteria, or we can stab a needle
of bacteria into the tube to see if there are any microbes that can grow without air (anaerobic).
Also, Petri dishes will only keep fresh for 3 weeks and then they dry out. Tubes will last for a
long time in the refrigerator, and are useful for making stock cultures (pure cultures).
MICROSCOPE USE: The arm should face you as you lift it up. Hold with one hand under base,
and one hand on the arm, gently set on desk near edge, and then turn the arm away from you.
Loosen the screw on the eye pieces, swivel the eye pieces towards you, then tighten the screw a
little. Fold up dust cover and put it in the drawer.
PARTS OF A MICROSCOPE: The basic frame of the microscope consists of the base, stage,
arm, and body tube.
Find the substage adjustment knob (left and right side, under stage), also called the condenser
knob. What happens when you turn it? It raises and lowers the condenser. When you are done
with the microscope, leave the condenser in the highest position; this is called racking up the
condenser. Everyone point to this knob.
The oculars are the eyepieces. They magnify ten times and focus the image on your retina. There
are two oculars. Do not touch your eyelashes to them or they will get oil on them. To clean,
squirt alcohol or lens solution (small bottle with pink liquid) onto lens paper (not Kimwipes)
and wipe, then wipe with dry lens paper.
The oculars have a diopter adjustment ring (knob with ridges), with plusses, minuses, and a
zero on it. What happens when you move it? It moves the ocular up and down. The purpose of
this is to allow the left eye to be focused independently of the right eye.
The coarse adjustment knob is for the dominant eye focus. Then use the diopter ring to focus
for your non-dominant eye. If you still see two images, you have a convergence problem and
you’ll need to keep one eye closed. Everyone has one dominant eye. You may need to close
that eye when you want to use the pointer, if it is on the wrong side for you. Between the
oculars is a disc with numbers on it. This determines the interpupillary distance in mm
(distance between your pupils). Adjust it like binoculars to get the right width comfortable.
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EYE DOMINANCE
Take a piece of paper, folded into a tube lengthwise. Hold the tube 12” away from your face and
look at someone. Tell me what eye I am using. That’s my dominant eye. Do this now with your
partner and have them tell you which eye is dominant on you. We use both of our eyes, but one
more than the other.
One of your oculars has a pointer. When you are using the pointer, switch that ocular to the
dominant eye. To switch the oculars, just pull them out, make sure you don’t drop them. Don’t
leave them off long, or dust will get in. To turn the pointer, just turn the ocular or move the stage
so the specimen meets the pointer. Always use the pointer to show me things when you have a
question.
The revolving nosepiece holds the objectives. Practice turning nosepiece by the ring, not by
touching the objective lenses. Listen and feel for the objective locking into place. You need to know
the following about the four objectives: The scanning (red ring) objective is the shortest, and
magnifies 4x. To find total magnification, multiply the ocular magnification (they come in 10x and
15x, but we only have 10x) by the magnification of the objective. What is total magnification on
our microscopes with the scanning objective? Since the ocular is 10x, and the scanning objective is
4x, the total magnification is 40x. The low power (yellow ring) objective is 10x, total mag is 100x.
The high dry (blue ring) objective is 40x, total mag is 400x. The oil immersion (white ring)
objective is l00x, total mag is 1000x. Always start with scanning (red) in front. After you focus it,
progress to higher magnification. Don’t use the white oil immersion lens unless you are looking at
bacteria, and use a drop of oil.
Focusing: Two knobs, coarse adjustment and fine adjustment. Fine adjustment is smaller and
sticks out from the middle of the coarse adjustment knob. Make sure the red ring is in place, then
move the coarse adjustment knob. The coarse knob moves the stage a lot, and the fine knob moves
it a little. The distance between the stage and the bottom of each objective lens is called the
“working distance”. Always start with the scanning objective (red ring), since it is the only one
that can’t hit the slide on the stage and break the lens and the slide. Put a slide on the stage, all the
way back and in the right corner of the slide holder, with the clamps holding it in. Then make sure
the condenser is racked up. Move the X/Y knob (dangling on the right side of the stage) so that the
center of the slide lines up with the light. Then turn the coarse knob so the stage is racked all the
way up. Look at the slide, then lower the stage with the coarse knob until it comes into focus. Only
after that can you switch to the next power up (yellow low power). To focus from now on, ONLY
use the fine adjustment knob. You can NEVER touch the coarse knob unless the RED ring is in
place. To take the slide off, switch to red ring first, then use the coarse knob to lower the stage.
PARFOCAL: This term refers to the factory adjustment which means that once you are focused
with the scanning objective, you are focused with all of the objectives (except for fine adjustment
for minor corrections). If you lose focus, always go back to the scanning objective.
The CONDENSER takes light from the lamp and makes the rays into a point on the slide. There
is a light intensity knob on the left side of the bottom of your scope. Adjust it for comfort and
visibility. In front of the condenser is a lever: the iris diaphragm lever. Swivel that from left to
right; it opens and closes like the iris in your eye (pupil) to regulate the amount of light allowed
10
in. When changing from low to high power, it is necessary to OPEN the iris diaphragm;
increasing magnification requires MORE LIGHT.
The iris does the following four things:
a. Regulates light intensity
b. Contrast: (when iris is open, the contrast decreases)
c. Depth of field (when iris is open, only the foreground is in focus. When the iris is
closed, the depth of field increases and everything is in focus).
d. Resolution (sharpness of image). Resolution is best when iris is open all the way.
Different microscopes have different resolving powers. The resolving power of a
microscope is a function of the numerical aperture (NA) of the lenses and the
wavelength of light.
STAGE ADJUSTMENT KNOB is on the right side, hanging down. It moves the stage forward,
backward, right, left. It is also called the x-y stage knob.
BE ABLE TO MATCH THE PARTS OF A MICROSCOPE TO A PHOTO OF A
MICROSCOPE FOR THE EXAM.
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GETTING TO KNOW YOUR MICROSCOPE
1. What is the working distance?
The working distance is the length (mm) from the objective lens to the slide. Since you start with
the lowest power (the shortest objective), as you increase magnification, working distance
decreases because the objectives are taller as you go up in magnification. The scanning objective
has the largest FIELD OF VIEW and the greatest working distance. When you are focused with
one objective, you are focused with all of them. What is this called? PARFOCAL.
2. What are the functions of the parts of the microscope in italics? Know the four functions of the
iris diaphragm (resolution, contrast, brightness, depth of field). Substage adjustment knob
(moves condenser up and down – when down, there is poor resolution). Diopter ring (allows left
eye to focus independently. Start with stage up, then bring down to focus the dominant eye.
Why does an image appear upside down and backwards? To answer this, you will look today at
the letter “e” at 40x, 100x, and 400x.
FOCUS SEQUENCE
1. Stage should be racked down.
2. Scanning objective should be in place.
3. Open stage clips (spring loaded).
4. Mount slide onto stage with specimen approximately centered.
5. Move stage all the way up with coarse adjustment knob.
6. Turn power on.
7. Adjust your chair so you are not bending.
8. Increase voltage until you see some light, but stay below maximum.
9. For most slides you need high contrast, so open the iris to the maximum resolution. When
you look at live cells, close the iris to decrease resolution.
10. Adjust oculars for the interpupillary distance until you see just one circle of light called
the FIELD. The type of microscope we have is a Brightfield.
11. Turn coarse adjustment knob to lower the stage slowly while looking through ocular for
the image to appear.
12. Make sure the specimen is still centered.
13. Close your left eye and use the coarse adjustment knob to focus the right eye.
14. Close your right eye and use the diopter ring on the ocular to focus the left eye.
15. Use the revolving nosepiece to move to the 10x objective (yellow). What happened to
your working distance? What happened to your field of view?
16. Image brightness will now decrease, so turn up the voltage control if needed.
17. Adjust the image contrast with the iris diaphragm lever.
18. Use the fine adjustment knob to focus. DO NOT TOUCH THE COARSE
ADJUSTMENT KNOB AGAIN! If you lose sight of the specimen, go back to the
scanning objective.
19. When finished observing at that power, turn revolving nosepiece to 40x (blue ring). You
will see a decrease in depth of field. If you are looking at a live specimen in water, you
will need to use the fine adjustment knob to follow it around.
20. Adjust for image brightness with voltage.
21. Adjust for contrast with iris. You can use the x/y adjustment knobs to move the stage
around if needed.
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Problems with the light on the microscope?
When the light does not come on, turn up the brightness of the light with the toggle on the
bottom left of the microscope until it is at full power. There will be a delay but it should work.
First make sure that the light source is turned up all the way before dimming it for viewing.
Sometimes the microscopes just need to be powered on all the way before the bulb starts.
Secondly check the breaker on the socket where the microscope is plugged in. The reset button
should be pushed in and a green light should be on. If the switch isn’t like this then the power
isn’t on to the switch. Push the reset button in all the way to fix this problem.
Take only one slide at a time. When done with a slide, bring it back and get another.
SLIDES TO LOOK AT NOW
1. Letter “e”
2. Blood smear. (look at RBC, WBC)
Practice moving the slide back and forth.
Why did the letter e look upside down and backwards?
The image gets bent as the light rays get bent through the lens, which is biconvex. Light entering
top and bottom of lens gets bent (refraction). This is caused because speed of light is changing.
As they travel through air, light rays go 186,000 miles per second. When they bump into some
glass, they slow down and get bent. Lenses allow us to magnify the image by focusing the light
rays at a point. As we move image back and forth, it allows us to focus. Then it hits mirror and is
sent to oculars and is bent again with your eye lens. Wind up with a focal point. That’s why
when you move slide to left, appears to go right. Our eye lens bends things upside down and
backwards; brain learns to switch the image. No brain in the microscope.
Under hi-dry look at RBC (pink) and WBC (purple nuclei). There are not as many WBCs as
RBCs. Practice putting the pointer on a structure and draw and label a picture of the WBC with
cell membrane, nucleus, and cytoplasm. Label the RBC with just cell membrane and cytoplasm
because there is no nucleus.
BACTERIA SLIDE
Bacteria are small, so they always need 1000x to be seen. The oil immersion lens has the
smallest depth of field. Start at 40x as usual, and the bacteria will just look like little dots. As you
progress to 100x, the dots are just bigger; focus again. At 400x, you will start to see shapes. The
details are not seen until you get to the oil immersion lens. Why is it called an oil immersion
lens? It needs a drop of synthetic immersion oil. Move the revolving nosepiece to half way
between 40x (blue) and 100x (white). Put a drop of immersion oil on the slide. When you rotate
the immersion oil lens into place, it will be immersed in the oil. Now there is no air between the
slide and the lens. Swivel the white ring into place.
There are three basic shapes of bacteria:
1. Spiral
2. Cocci (singular is coccus)
3. Bacilli (singular is bacillus; also known as rods)
Some slides have all three shapes. Make sure you see all three today.
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CLEANING UP OIL
The oil immersion lens has a sealer around it so the oil cannot seep in, but the other objectives
are not sealed, so don’t get oil near them.
1) When you are done with the oil (white) lens, swivel the nosepieces toward the RED ring
(not the blue). If you swivel to the blue, the oil will get in the blue objective and ruin it.
2) Once on red, lower the stage, and use half a piece of LENS PAPER to DAB the oil from
the white oil lens. Dab it on a clean spot on the lens paper then dab again at another clean
spot on the lens paper, and continue to dab until no more oil is coming off.
3) Then take the other half of the lens paper and apply a few drops of alcohol (in the pink
bottle on the lab bench shelf). Use this to dab on the oil lens several times again.
4) Then use a clean lens paper with alcohol to dab the blue lens as well to be sure it is clean.
5) Then get a Kimwipe (not lens paper) to clean the oil off the slide.
6) Then get a Kimwipe to clean the oil and fingerprints off the stage.
7) Then use a Kimwipe and alcohol to clean the oil off the condenser and any other parts of
the microscope.
How to Put the Microscope Away
1)
2)
3)
4)
5)
6)
When you are done with the microscope, turn the light intensity all the way down.
Then turn the power switch off.
Then unplug the microscope.
Make sure the 4x (red ring lens) is in the center viewing position.
Use the coarse adjustment knob to put the stage all the way down.
Use LENS paper to clean off oil from the oil (white) and high dry (blue) lenses. If they are
very oily, use the pink alcohol cleaner on the lens paper.
7) Use KIMWIPES to remove oil and fingerprints from the stage and the rest of the
microscope, using the bottle of pink oil cleaner on the top of the lab bench.
8) Make sure the Eyepiece Oculars are turned to storage position (with the viewing end
facing the back of the microscope) AND screwed down. If the Eyepiece is facing the
front of the microscope there is a chance that the oculars could hit the back of the
cabinet if pushed in hard enough. If the eyepiece is not screwed down then there is a
change that the ocular area could be damaged as the cover is pulled off the microscope.
9) Put the dust cover on.
10) Make sure your microscope goes back into the cabinet with the same number as the
microscope.
11) When the microscope is placed in the cabinet, make sure the handle for carrying the
microscope faces the next user so they can pick it up by the arm.
12) Make sure the cord is not wrapped around the Microscope in any way. Please have the
cord placed loosely beside the microscope after it is placed in the cabinet.
13) Do NOT put your lab coat in the Microscope cabinet. It contaminates the microscope
covers.
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