Micro Lab Test

Question 1

Donning (putting on/entering)

Answer 1

-place books on disinfected area.
-lab coat
-goggles
-gloves
*ensure hair is tied, no jewlery, phones, exposed skin.

Question 2

Doffing (taking off/ exiting)

Answer 2

-remove gloves with proper procedure
-goggles
-lab coat
-wash hands

Question 3

Lab Bench

Answer 3

use a copious amount of cleaner (Vanguard disinfectant) to disinfect the bench. make sure to get the edges.

place the material brought into the second drawer beside the work station.

if working with bacteria place a rack on the left of your work area if you are right-handed and the opposite if you are left-handed.

Question 4

Aseptic Technique

Answer 4

ensure no lids or tools are placed on the bench. take the lids off with your pinky and retrieve bacteria with your pointer finger and thumb.

Question 5

Incineration

Answer 5

turn it on 10 minutes before the procedure.

tools used for bacteria are incinerated for ten seconds between different bacteria to avoid mixtures.

Question 6

Bunsen Burner

Answer 6

Operate a Bunsen burner by turning on the gas, igniting the flame with a striker, adjusting the air hole for a blue flame, and turning off the gas when finished.

Question 7

Bright Field Microscope SOP
1)Setting up
2)Setting up 100X
3)Putting Away

Answer 7

Setting up
-Remove cover from the microscope and place in the third drawer on your right.
-place microscope in the center of your work station.
-ensure microscope is off and plug in.
-obtain slide from front bench
-gently lower position with coarse adjustment knob.
-place slide label up, put into 10X, raise stand to highest point, place iris diaphragm to 10.
-put light to 3X using on switch.
-use fine adjustment knob to focus.
-place the object of interest into center.

Setting up 100X
- twist revolving nose piece counterclockwise until 10x is slightly out of position.
-add 1 drop of oil immersion, turn to 100X without going through 40X, adjust iris diaphragm to 100.
-focus with fine adjustment knob.
-observe and draw.

Putting Away
-lower stage with coarse adjustment knob.
-remove slide and wipe with kim wipe.
-gently wipe 100X with lens paper. verify 40X is not contaminated.
-return 10X back into first postion.
-turn off, unplug, cover.
-place slide back in the front.

Question 8

Bacillus subtilis (Shape, Arrangement, Gram Staining?)

Answer 8

1) Large Rod
2) Cluster
3) Violet Gram ve+

Question 9

Staphylococcus epidermis (Shape, Arrangement, Gram Staining?)

Answer 9

1) Cocci
2) Cluster
3) Violet Gram ve+

Question 10

Escherichia coli (Shape, Arrangement, Gram Staining?)

Answer 10

1) Small Rod
2) Cluster
3) Red Gram ve-

Question 11

4 Flagella Locations

Answer 11

1)Monotrichous- one on the right side
2)Lophotrichous-multiple on the right side
3)Amphitrichous- one on each side
4)Peritrichous- Like hair all over

Question 12

Bacterial Smear

What is the element you are trying to achieve?

Answer 12

-Add one loopful of water to the slide with a sterile loop.
-Add a loop of bacteria to the slide.
-Smear in a circular oval motion.
-Heat fix the bacteria to the slide using the bacti-incinerator or Bunsen burner.

We try to achieve a monolayer with a bacterial smear.

Question 13

Why is gram staining so important to field microbiology?

Answer 13

Gram staining is crucial in microbiology as it categorizes bacteria into Gram-positive and Gram-negative groups, guiding treatment decisions, aiding in bacterial identification, and playing a key role in infectious disease diagnosis.

Question 14

How is gram staining done? Function of each solution and time?

Answer 14

1) Crystal Violet (1min)- Primary stain
Rinse in H2O
2)Iodine (1min)- Mordant, CV-I complex(binding) with Crystal violet
Rinse
3)Alcohol (15sec)- Decolourizer
Rinse
4)Safranin (30 sec)- Counterstain, if negative it will stain red
Rinse
place on blotting pad of paper

Question 15

Why does envelope of gram + stain purple?

Answer 15

Gram-positive bacteria look purple after staining because the crystal violet dye sticks to the thick peptidoglycan layer in their cell walls (does not allow solvent to penetrate them), creating a purple color during the staining process.

Question 16

Envelope(gram+) vs Envelope(gram-) structural difference?

Answer 16

Envelope +: 1) Contains a cell wall with thick peptidoglycan and has a tetrapeptide side chain. stains violet.
2) Contains plasma membrane with protein surrounding the phospholipid.

Envelope -: 1) Contains outer membrane with protein and pore surrounding Lipopolysaccharides (O-antigen and Lipid A).
2) Contains periplasm with lipoprotein and peptidoglycan
3)contains plasma membrane with phospholipids and protein surrounding.

Question 17

Why do cells stain variable?

Answer 17

older than 24hrs, thick layer on bacterial smear, and mixed culture.

Question 18

Simple stain (general shape)

Answer 18

-after slides are prepared and heat fixed place in one dye either safranin, crystal violet, or methylene blue for 1 min.
they should all be gram +

Question 19

Gram stain results (shapes, arrangements, +or-)

Answer 19

Cocci
1)pairs (diplococcus)
2)chains (streptococcus)
3) tetrad cocci (four)
4)cluster (grapelike- staphlococcus)
5)cubical packets of 8 (sarcina)
6)singles (no arrangement)

Bacilli
1)pairs (diplobacillus)
2)chains (streptobacillus)
3)side by side (palisading)
4) snapping v
5) singles
6) 1/2 rod and cocci (coccibacillus)

Spirlla
1)one half-moon bean shape (vibro)
2) loosely wound spiral (Spirillum)
3)tightly wound (spirochete)

Question 20

Capsule stain (we used Klebsiella pnuemoniae- resistant to antibiotics)

Observes the capsule protecting the bacteria

Answer 20

• 2 loopfuls of bacteria are mixed into a small drop of india ink
  -spread over slide by placing the cover slip at the top and pull down
  -air dry then heat fix
  -counter stain in crystal violet for 1 min
  -rinse and examine with oil immersion objective
  -rod will be purple and capsule will be clear

Question 21

Negative stain (we used staphylococcus epidermis)

to observe shape of cell, arrangement: background was black

Answer 21

-drop nigrosine at the end of the slide closest to the label
- 2 loopfuls of bacteria are dispersed in a small drop of nigrosine
-spread by placing the cover slip at the bottom, move up past nigrosine, and drag to the bottom
-air dry do not heat fix
-examine using oil immersion

Question 22

Spore stain( to stain spores within each cell so we can see the spores)

Answer 22

-prepare fixed bacteria smear
-place smear on heating tray and cover the slide with absorbent paper to prevent drying
-pour malachite green stain over paper until the smear is flooded than heat underneath with bunsen burner until dye steams
-keep dye steaming for 6-7 mins (dont let dry out keep on adding dye)
-after slide has cooled rinse to get paper off and dye
-place slide into safranin for counter stain 1min and rinse
-use blotting paper to dry and examine under oil immersion lens

Question 23

what is streaking for isolation based on?

Answer 23

based on the premise of diluting the bacteria out on the plate by streaking them in a specific fashion.

Question 24

why is it important to have a pure culture?

Answer 24

Having a pure culture is important because it allows accurate identification and study of specific microorganisms without interference, ensuring reliable results.

Question 25

what does streaking for isolation appear like on a plate? how do you do it?

Answer 25

Streaking for isolation on a plate typically appears as a pattern of bacterial colonies that gradually become more spaced out, helping to isolate individual bacterial cells and obtain pure cultures for further study.

spread culture back and forth in one small sections, incinerate, two streaks from the small section (do to cross over), continue (do not incinerate again) only go through once with zigzag motion, continue till the plate is covered with no overlap.

Question 26

Colour/Reaction of…
1) Carbohydrates (Glucose, Lactose, Sucrose)?

2) Triple Sugar Iron?

Answer 26

1) yellow(acid)+, red(alk)-, gas production circled, reversion (red top yellow bottom)

2)-Red slant yellow butt (alk/acid): only glucose)
-Yellow slant yellow butt (acid/acid): lactose and or sucrose with addition to glucose
-Red slant red butt (alk/alk): no fermentation only peptone
-H2S: Black colour on the butt, when it is combined with ferrous sulphate

Question 27

Colour/Reaction of…
1) Urease
2)IMViC (indole, Methyl Red, Vogues: Proskauer, Citrate)

if MR is + why do we know Vp is -?

3)Motility and Malonate

Answer 27

1)Urease: Red/ Pink +, Yellow -

2)-Indole: Red ring+, No RR-
-Methyl Red and VP: red(pH6-7) +, yellow(pH4-5)
-Citrate: blue+, green (no growth)

Because MR is always opposite from VP

3)-Motility: fuzzy red +, clear -
-Malonate: blue+, green- no growth

Question 28

Colour/Reaction of…
1)Decarboxylase (Lysine, Arginine, Mio- Ornithine)

why is it so important not to shake the tubes during transport?

Answer 28

1) -Purple: +, yellow -
-Purple top/ yellow bottom: -
-Greyish colour: delayed positive
-Purple top/ yellow middle/ purple bottom: +

It is important not to shake the tubes during transportation due to risk of mixing the colours, making it hard to identify the tests

Question 29

What does reversion mean?

Answer 29

Reversion is when something that changed goes back to the way it was before. In genetics, it means a mutation or alteration returns to its original form.

Question 30

Colour/Reaction of…

1)Gelatin @ 21degrees celcius
2)Phenylalanine
3) Nitrate and Zinc

Answer 30

1)Gelatin: solid medium -, Liquid medium: +
2)Phenylalanine: green +, no colour change-
3)Nitrate: red+, no colour change- (add zinc, if red-true positive, no colour change+)

Question 31

Colour/Reaction of…

1)Catalase
2)Oxidase

Answer 31

1)Catalase: add hydrogen peroxide to a slide than bacteria, bubble gas production +, no gas production -.

2)Oxidase: place on a strip if blue +, no colour change -

Question 32

Algae Lab (Pro or Euk, Cyanobateria or Protozoa or Algae, Group?

1) Anabaena?
2) Peridinium?
3) Spirogyra
4) Volvox
5) Diatoms
6) Clorella

Answer 32

1) Pro, Cyano, Cynobacteria
2) Euk, Protozoa, Dinoflagellates
3) Euk, Algae, Unicellular Green algae
4) Euk, Algae, Unicellular green algae
5) Euk, Protozoa, Dinoflagellates
6) Euk, Algae, unicellular green algae

Question 33

Fungi Lab (review lab)

Answer 33

idk what to put

Question 34

Differential Media (DM)-gram stain, shape and arrangement, MacConkey, and Salt Mannitol?

Escherichia coli?

M.Morganii?

Answer 34

E.coli: -, Rod/Cluster, LF (Colonies appeared pink), did not grow on SM

M.Morganii: -, Tiny rod/ cluster, NLF (colonies appeared beige, did not grown on SM

Question 35

Differential Media (DM)-gram stain, shape and arrangement, MacConkey, and Salt Mannitol?

S. aureus?

S. epidermis?

Answer 35

S. aureus: +, Cocci/Cluster, did not grow on MAC, MF (yellow halo)

S. epidermis: + Cocci, cluster, did not grown on MAC, NMF (no yellow halo, red)

Question 36

The selective components of MacConkey Agar are crystal violet and bile salts which eliminate Gram “__” ve bacteria. _______salt also inhibits the growth of non-enteric bacteria. The differential component in MacConkey Agar is the sugar, ____________. When this sugar is fermented, it produces an ________condition. This changes the color of the pH indicator known as ___________ to a _____color. The bacterial colony turns the same color. Bacteria that do not ferment this sugar can use peptone in the medium, producing an alkaline reaction and
_____________ colored colonies.

The selective component of Salt Mannitol Agar is 7 – 10% _______. This inhibits the growth of bacteria that do not tolerate a high level of this chemical.

The differential component of Salt Mannitol is the sugar _____________. When this
sugar is fermented, _________ is produced. This alters the pH indicator called _____________ and changes it to a __________ color. The bacterial colony and the
surrounding medium turn the same color. Non-fermenting bacteria, which use the peptone in the medium, do
not produce any change in the color of the colonies and its surrounding medium.

Answer 36

The selective components of MacConkey Agar are crystal violet and bile salts which eliminate Gram “+” ve bacteria. ____BILE____salt also inhibits the growth of non-enteric bacteria. The differential component in MacConkey Agar is the sugar, _____LACTOSE_______. When this sugar is fermented, it produces an ____ACIDIC ____condition. This changes the color of the pH indicator known as ______NEUTRAL RED___ to a ___DEEP PINK__color. The bacterial colony turns the same color. Bacteria that do not ferment this sugar can use peptone in the medium, producing an alkaline reaction and ___BEIGE___ colored colonies.

The selective component of Salt Mannitol Agar is 7 – 10% __SODIUM CHLORIDE____. This inhibits the growth of bacteria that do not tolerate a high level of this chemical.

The differential component of Salt Mannitol is the sugar _____MANNITOL_____. When this
sugar is fermented, ___ACID__________ is produced. This alters the pH indicator called ____PHENOL RED______, and changes it to a __YELLOW_____ color. The bacterial colony and the surrounding medium turn the same color. Non-fermenting bacteria, which use the peptone in the medium, do
not produce any change in the color of the colonies and its surrounding medium.