Micro Lab

Lab PPE

1) lab coat

2) hair tied (long)

3) get rid of jewelry

4) closed toe shoe

5) gloves

Sanitation desk & handwashing & microscope (when & how)

Handwash

• before/after lab wash hands

a) cold & hot faucet on

b) rinse hands in water

c) lather soap under nails and hands

d) rinse

e) paper towel & wipe

f) use paper towel to turn off sink

g) dispose paper towel

Desk

• before/after labs clean desk

a) drench lab table in bleach; leave a film

b) wipe

Microscope

• before/after use is ideal

a) wipe lens w kim tech wipes & cleaning sol → get rid of oils & stuff on screen

b) lower stage after ALWAYS

Personal safety FOOD & storage

1) Do not eat, chew gum or drink in lab

2) electronics & class work material (lab, workbooks) must be stored at safe area w/ little contamination

Disposal

• hazardous material

• glass

• test tubes

• contaminated waste w/body fluids

• nonbiohazard

Disposal

1. no HAZARDOUS & infectious material in sink (like: MATCH & PAPER)

2. GLASS disposal in hazardous container

3. test tubes→ remove label→ dispose in proper container

4. contaminated waste w/body fluids→in proper container

5. non biohazard→ in designated containers

Spill (keep in mind glass and spill)

1. Pour disinfectant

2. Inform professor and students

3. Let 10 mins pass

4. Use paper towels to soak

5. do NOT use bare hands (gloved) to get towels if glass SWEEP w broom

Aseptic technique & quadrant

1. Sterilize WHOLE inoculating loop w flame

2. let it rest on metal

3. open test tube (pinky method) & heat lip

4. grab loop put it in once all the way

6. flame lip & seal tube w cap (pinky method)

7. but loop in medium ( agar or liquid)

8. 1st quadrant

9. strelize loop

10. DRAG FROM FIRST to 2nd quadrant

11. sterilize

12. DRAGE FROM SECOND to 3rd quadrant

13. sterilize

14. DRAG FROM THIRD to 4th quadrant

15. sterilize

16. seal medium

1. Fire alarm

2. fire extinguisher

3. eye wash

4. shower

5. first aid kit location

1. door of entry

2. near door of inner office

3. near door of inner office

4. near door of inner office

5. on shower & wash station wood

If fire starts or fire alarm sounds ( 3 things)

• unplug electrical apparatus & turn off burner

• press button at door of inner office to shut off gas

• evacuate in orderly manner

if you have contact lenses and something gets into ur eye should you take it off?

• no, lenses can melt onto eye and don’t want to peel it and ruin eye

Holds the objective lens and can be rotated to change the magnification.

Rotating/Revolving Nosepiece

Eyepiece which contains a lens to magnify an image 10x that projected by the objective lens.

Ocular Lens

Connects the Ocular Lens to the Objective Lenses and provides alignment to direct the light from the specimen into the viewer's eye.

Body Tube

Connects the body tube to the base. One hand should be around this when carrying a microscope.

Arm

First knob you should use and always under low power, makes large adjustments to the focus. Should never be used in high power.

Coarse Adjustment Knob

The second knob you should use under higher power for exact focusing.

Fine Adjustment

Adjustable lens system that permits the use of a low power lens, a medium lens, or a high power lens

Objective Lenses

This objective lens provides the lowest magnification, 4 x

Scanning Power

This objective lens provides a medium magnification, or power, 10x

Low power

This objective lens provides the abt the highest magnification, 40x

high power

Objective used w/ oil 100x

• Oil-immersion

Where you place the slide containing the specimen; It contains a hole that allows light to pass through the stage and onto the specimen.

Stage

Holds the slides in place on the stage for viewing.

Stage Clips

It contains a dial that rotates to adjust the amount of light that reaches the specimen and is passed upwards towards the eyepiece. Change it w what?

• iris diaphragm

• change w obj lense power

Supports the weight of the microscope. Contains the electronics and the light source.

• Base

An electric source of illumination or a mirror used to direct light upward.

Light Source/ illuminator

The lens under the stage that focuses light from the illuminator through to the hole in the stage

Condenser Lens

Total Magnification =

Ocular Lens Magnification x Objective Lens Magnification

As obj lense mag (+)

• FOV decreases

microscope inverts

image vertically and laterally

Why do we use oil only with the 100× objective?

• oil = light control (bends and travels) for high power only.

Why do we calibrate the ocular ruler at different total magnifications bc each lense has what and allows which conversion?

ensures accurate measurement of microorganisms bc→

a) each objective lens has a different µm-per-ocular-unit value

b) allowing conversion of ocular units into real metric units.

Know ur values

Objective	Total Magnification	Millimeters (mm)/ space	Micrometer (Mm)/space
4x
10x
40x
100x

Objective	Total Magnification	Millimeters (mm)/ space	Micrometer (Mm)/space
4x	40x	0.025	25
10x	100x	0.01	10
40x	400x	0.0025	2.5
100x	1000x	0.001	1

does cell shape change with objective lense changing

• No, ONLY lenses CHANGE

Of a cell is 20 ocular spaces in 40x total mag what is micro or milimeter?

• 0.5mm

• 500 micrometer

when is plate properly streaked?

1. describe the amount of growth in FIRST area

2. describe growth in later quandrants

3. where should isolated colonies be

• Heavy growth in the first streak area

• Progressively lighter growth in each subsequent streak

• Well-isolated colonies in the final streak area

A plate is not properly streaked if you see:

• growth where?

• isolated or no isolated___?

• which kind of streaks?

• overlapping?

• Growth covering the entire plate (no dilution)

• No isolated colonies

• Thick, continuous streak lines

• Colonies overlapping or merging together

How does chromogenesis help distinguish microorganisms?

• allows microorganisms to produce different colored colonies on chromogenic media →making it easy to visually differentiate species in mixed cultures.

when is something a pure culture?

• size, shape, texture, & color are consistent throughout

can you isolate a single microbe from a mixed culture?

• Yes→streak method→separate individual cells→isolated colonies

6 forms known as what?

1. Circular

2. Spindle (diagonal oval)

3. concentric (target)

4. irregular

5. filamentous (filament like)

6. Rhizoid (like filamentous but less complex)

6 Margins look like what?

1. Entire (circle)

2. undulate (slightly spiky)

3. scalloped (looks like scallop w ridges)

4. erose (pom pom)

5. lobate (nickelodeon drop)

6. filamentous (same)

6 elevations

1. flat

2. raised

3. subsurface (underneath)

4. convex (contact lenses)

5. pulvinate (lid on globe)

6. umbonate (bottle cap)

4 Appearance terms

1. dull

2. glistening

3. shiny

4. wet-looking

4 optical properties (TIOO)

1. opaque

2. opalescent

3. iridescent

4. translucent

Pigmentation 2 types & know what they mean

1. pigmented

2. nonpigmented

5 surface texture types?

1. Smooth

2. wrinkled

3. rough

4. powdery

5. dry

Microbe HUNT→How were nitrogen-fixing microbes enriched from soil?

By culturing soil in a nitrogen-free medium, so only microbes that can fix atmospheric nitrogen grow.

MICROBE HUNT→Thermoduric treatment

• heat treatment killed NON-heat-resistant bacteria while allowing heat-resistant or spore-forming bacteria survive & grow

Microbe HUNT→How were benzoate-using microbes enriched from soil?

By using a medium where benzoate is the sole carbon source, selecting for microbes that can metabolize benzoate.

MicrobeHUNT→Why use enrichment instead of streaking soil directly onto nutrient agar?

Enrichment selectively favors target microbes and prevents fast-growing, non-target organisms from dominating growth.

Bacteria (NOT EUKARYOTES→Yeast, Paramecium, & Mold)

A) 3 shapes known as what?

B) Within those shapes, which has 6? Which has 5? Which has 3? All of them known as what?

C) which one is grape shape? which one is rice shaped? which one is spaghetti shaped?

D) which one is in between cocci and bacillus?

A) Coccus (circle)

1. coccus (one grape)

2. diplococcus (two sets of grapes)

3. streptococcus (string of grapes)

4. tetrads (one cube layer of grapes)

5. staphylococcus (cluster of grapes)

6. sarcinae (layered cube of grapes)

B) Bacilli (rod-shaped)

1. Bacillus (one rice grain)

2. diplobacillus (2 rice grain lined)

3. streptobacillus (4 rice grain lined)

4. palisading (4 rice grain cube)

5. snapping (2 rice grain V shape)

C) Spirlia (spiral)

1. vibrio→ short pasta

2. spirillum→ soft curly spaghetti

3. spirochete→corkscrew spaghetti

D) coccobaccilli

1st step Crystal Violet (Primary stain)

1. what color?

2. # stain applied?

• Colors all bacteria purple

• First stain applied

2nd step Gram’s Iodine (Mordant)

1. forms what type of complex is it

2. helps what

• Forms crystal violet–iodine complex

• Helps the dye stick inside gram-positive cells

3rd step Ethanol or Acetone (Decolorizer)

• Which step?

• Removes dye from which bacteria?

• Which retains color?

• Differential step

• Removes dye from gram-negative bacteria

• Gram-positive bacteria retain purple color

4th step Safranin (Counterstain)

• what color stain for which type of cells?

• which type of cell remains which purple?

• Stains gram-negative bacteria pink

• Gram-positive bacteria remain purple

Why does gram (+) retain dye versus gram (-)? Because of this difference what color do they remain?

• Gram-positive bacteria have a thick peptidoglycan layer

• Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane

because of this…

• Gram-positive bacteria retain crystal violet → purple

• Gram-negative bacteria lose crystal violet and take up safranin → pink

simple v.s. negative v.s. differential stain

• Simple stain: one dye, all cells the same color

• Negative stain: stains the background, not the cells

• Differential stain: uses multiple dyes to distinguish cell types

If the Gram’s iodine step was skipped, what color would the cells likely be?

Most cells would appear red/pink.

Why:
Gram’s iodine forms the crystal violet–iodine complex.
If iodine is skipped:

• Crystal violet is not fixed in the cell

• Ethanol removes it from both types

• Safranin stains most cells pink

what stain for flagella?

flagella stain is required.

Introducing a sample into a nutrient medium is called:

Inoculation = placing microorganisms into a growth medium.

Transferring a sample to a separate medium to obtain a single microbe (pure culture) is called:

Isolation separates individual microbes so a pure colony can grow.

Macroscopically observed colony characteristics include all EXCEPT:

Cell shape requires a microscope

What is the most universal diagnostic staining technique for bacterial identification and classification? & what type?

• Gram stain

• Differential stain

  Why:
  It uses multiple dyes and produces different colors depending on bacterial structure.

In addition to liquid, which are physical types of media?

• know what each is used for or what they contain

• Solid (convertible to liquid) → contains agar

• Solid (not convertible to liquid) → egg-based media

• Semisolid → used to test motility or oxygen requirements

5 i’s

1. Inoculation – placing the specimen into a growth medium

2. Incubation – storing the culture under proper conditions so microbes grow

3. Isolation – separating individual microbes to obtain pure colonies

4. Inspection – observing colony morphology and microscopic features

5. Identification – determining the exact microorganism (stains, tests, etc.)

The Gram stain follows a specific sequence (four) :

1. Crystal violet (primary stain)

2. Gram’s iodine (mordant)

3. Alcohol or acetone (decolorizer)

4. Safranin (counterstain)

Why use heat with certain stains and not with other ones?

• Use heat for structures that are tough and impermeable (endospores, acid-fast walls).

• Avoid heat for fragile structures (capsules, some glycocalyx or flagella stains).

Why use basic dye? Why fix cells? And why rinse with water?

Use basic dye

Positively charged → binds negatively charged cells

Fix cells

Kill, stick to slide, preserve structure

Rinse with water

Remove unbound dye, improve contrast

3 reasons why is it a bad idea to throw out shards of glass and paper towel in normal trash?

1. glass could injure custodial staff or others

2. glass is most likely contaminated with microorganisms

3. biohazard material like the towel needs to be specifcally disposed to prevent infection and exposure

why should 4x and or 10x be used to intially focus on specimen

1. large FOV→easy to focus & find specimen→doesn’t hit glass slide

What is chromogenesis, and how can it be used to define the purity of culture? What if contaminated?

1. bacteria→pigment→clarity of morphology

2. size, texture, shape, elevation, and color will not resemble one another

Can a pure culture containing a single type of microbe be prepared from a culture with a mixture of cells explain?

Isolation on solid media + colony selection → pure culture from a mixed culture.

Eukaryotic cells are distinguished from prokaryotic cells by the presence of internal membranebound structures called organelles. In which eukaryotic cell can you find internal structures? What do you think these structures are?

(bacillus, anabaena (green), saccharomyces/yeast (yellow), aspergilus niger/mold (yellow green))

• eukaryotic cells (Saccharomyces (yeast) & Aspergillus (mold) ) have internal membrane-bound organelles. Prokaryotes rely on cytoplasmic structures, not true organelles

We add immersion oil to view bacterial slides.Why is the oil NOT used with the Paramecium slide?

• Use oil immersion: for very small specimens (bacteria, fine structures) under 100× objective.

• Do NOT use oil: for large eukaryotic microbes (Paramecium, amoeba, yeast) viewed at 10× or 40×.

What happens if you accidentally flip your slide upside-down so the specimen is on the bottom? Can you still see it clearly with different objective lenses?

Specimen may be visible but blurry; low/medium power might work with refocusing, high-power/oil won’t focus properly.

Why are thick or dense smears less likely to produce a good smear preparation for microscopic evaluation?

Too many cells overlap → hard to see individual cells and their shapes.

Why is it essential that smears be air-dried rather than gently heated over a flame to speed up the drying process?

Heat can distort or burst cells; air-drying preserves shape for staining.

Why is fixing important during the direct staining process?

Fixing kills cells, sticks them to the slide, and preserves their shape.

Which part of the cell is a basic stain actually staining

The negatively charged cell surface (membrane, wall, cytoplasm).

Think about what these biological stains are doing, and how they "stick" to the cells even when rinsed with water.

Do you think that coffee would be considered a good biological stain?

After all ifyou spill it on a white shirt, the shirt will have a coffee stain. (HINT: A coffee stain that is still wet will rinse out with just water.)

No. Coffee rinses away with water → it doesn’t bind to cells like a proper dye.

When doing 5 I’s with colony v.s. culture what is different, exceptic ASEPTIC technique

1) colony→ water on slide→inoculate & mix → fix→stain→rinse

2) culture→inoculate driectly on slide→fix→ stain→rinse

How does a basic dye differ from an acidic dye? (HINT: Think about differences in the chemicals and what they can Interact with).

1) basic dye (+)→(-) of a cell like cell wall & DNA

2) acidic dye(-)→(-) of a cell=repelling→ doesn’t stick to cell but background

When you perform a negative stain, why does the specimen appear light, while the background is stained dark?

1. (-) indirect dye repels (-) part of cell→stains background

When you perform a simple stain on cells, why does the direct stain (with a basic dye) require rinsing with water, while an indirect stain (with nigrosin) does not?

1. direct stain (+)→ sticks (-) of cell→rinsing is NEEDED to get rid of extra dye→aid in visibility of cells

2. indirect stain (-)→ repels cell→ sticks but not as tight as direct→ rinsing would wash the stain off→ which is a NO→ since we need it to see shape of cells (CAN’t see inside though)

Which method, direct or indirect staining, do you think would give you a better estimate of the true size of a microorganism? Explain your reasoning. HINT: Bacteria cells can have structures outside of the cell wall layer.

1. indirect (-)→ doesn’t stick or need heat fixation→ which will alter cell’s shape & will visualize all structures outside → for accurate size determination this method is recommended