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MICROBIO BIOL-160 LEC > Lab Practical Review > Flashcards

Lab Practical Review Flashcards

1
Q

definition of a BACTERIAL COLONY

A
  • where bacteria begins to grow on a SOLID SURFACE
  • COLONY: a VISIBLE MASS of BACTERIA–arising from SINGLE BACTERIAL CELL
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2
Q

what characteristics are in COLONY MORPHOLOGY (7)?

A
  • SIZE (colony dimensions)
  • SHAPE (round, irregular, punctiform)
  • MARGINS ( entire, lobate, undulate, rhizoid)
  • SURFACE (smooth, rough, wrinkled, shiny, dull)
  • TEXTURE (moist, mucoid, dry)
  • ELEVATIONS (flat, convex, raised etc…)
  • OTHER (color, optical prop.)
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3
Q

what other FACTORS make bacteria grow?

A
  • temperature
  • time of growth
  • nutrient avalibility
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4
Q

definition of MICROSCOPY

A

the field of USING MICROSCOPES to look at OBJECTS that cannot be seen with the NAKED EYE

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5
Q

what are the THREE WELL-KNOWN BRANCHES of MICROSCOPY?

A
  • LIGHT
  • ELECTRON
  • SCANNING PROBE
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6
Q

definition of COMPOUND LIGHT MICROSCOPE

A
  • has a SERIES OF LENSES
  • “COMPOUND”–magnification of various LENS–giving viewer an ENLARGED INVERTED IMAGE of the object
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7
Q

what are the OBJECTIVE and TOTAL MAGNIFICATIONS?

A

LOW POWER SCANNING:
4x - 40x

LOW POWER:
10x - 100x

HIGH-DRY:
40x - 400x

OIL:
100x - 1000x

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8
Q

condenser

A

condenses the light beams on the actual specimen

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9
Q

objective

A

where the light bend/refracts and creates magnified initial image

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10
Q

ocular lenses

A

magnifies the final image as virtual image

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11
Q

phase contrast microscope

A

use of a SPECIAL CONDENSER that BENDS LIGHT
- gives great CONTRAST of INTERNAL STRUCTURES

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12
Q

dark-field microscopes

A

use of a DARK-FIELD CONDENSER to BLOCK LIGHT from the OBJECTIVE LENSES–appears as LIGHT against DARK BACKGROUND

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13
Q

how do we STORE our MICROSCOPES?

A
  • lens always at the LOWEST OBJECTIVE
  • CORD is wrapped and secured
  • OCULARS pushed together
  • STAGE is all the way down
  • use BOTH HANDS to carry
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14
Q

what is important about EFFECTIVE HAND WASHING?

A
  • helps to MINIMIZE DIRECT PERSON to PERSON + INDIRECT CONTACT transmission of PATHOGENS
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15
Q

how does SOAP play a role in terms of handwashing?

A

has specific ACTIVE INGREDIENTS to either KILL (BACTERICIDAL) or STOP BACTERIA GROWTH (BACTERIOSTATIC)

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16
Q

definition of CULTURE and PURE CULTURE

A

CULTURE:
media that contains living microbes

PURE CULTURE:
contains only one species

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17
Q

definition of ASEPTIC METHOD

A

the transferring of MICROBES from pure culture to a STERILE CULTURE

  • avoiding any chance of CONTAMINATION to urself, others, and environment
18
Q

What are the types of media that we have?

A

BROTHS:
growing microbes in FRESH CULTURES are large numbers of cells are requirements

AGAR SLANTS:
Growing STOCK CULTURES that can be refrigerated after incubation and maintained for several weeks

PLATE MEDIAS:
Use for obtaining ISOLATION of species

19
Q

What are our transfer instruments?

A

Inoculating loops, or inoculating needles

20
Q

Describe procedure of ASEPTIC TRANSFER: BROTH PURE CULTURE to STERILE BROTH TUBE

A
  1. Label sterile tube with necessary details
  2. Use inoculating loop
  3. Flame and sterilize inoculating loop
  4. Flame and sterile broth tube
  5. Remove cap and insert loop—should have a film now
  6. Reflame tube and close
  7. Pick up sterile broth tube
  8. Flame sterile broth tube
  9. Remove cap
  10. Mix the INOCULUM into sterile medium
  11. Remove loop and reflame sterile broth tube (close)
  12. Flame loop
    DONE :)
21
Q

Describe ASEPTIC TRANSFER: AGAR SLANT CULTURE to STERILE AGAR SLANT

A

*remember sterilization methods and flaming of tubes each time when opening and closing

  • touch sterilized loop onto just the BASE OF THE SURFACE OF AGAR SLANT
  • once picked up, we go to our STERILE AGAR SLANT—use of the FISHTAIL STREAK 😨
22
Q

Definition of MIXED CULTURE

A

microbial culture that contains TWO OR MORE SPECIES

23
Q

Why do we use the ISOLATION TECHNIQUE on the STREAK PLATE?

A

helps to DECREASE CELL DENSITY and we can actually see INDIVIDUAL CELLS being deposited —can help grow them into COLONIES

  • can create a proper COLONY FORMING UNIT
24
Q

Quadrant streak method

A

quadrant streaks—sterilizing loop after first initial streak in quadrant to decrease cell density over time

25
Q

What is the SPREAD PLATE TECHNIQUE?

A

A method of ISOLATION —using a DILUTED MICROBIAL SAMPLE onto AGAR PLATE, spread with a glass rod

  • can help grow into INDIVIDUAL COLONIES
26
Q

Describe SIMPLE STAINS—what are CHROMOGENS and CHROMOPHORES?

A

STAIN:
has a solvent and specific COLORED MOLECULE (CHROMOGEN)

**gives color through CHROMOPHORES (has an AUXOCHROME which is CHARGED)

27
Q

How do AUXOCHROME AND BASIC STAINS INTERACT?

A

AUXOCHROME - positively charged
BASIC STAIN - negatively charged

are ATTRACTED TO EACH OTHER—why we have dyed stains

28
Q

What are our TYPICAL BASIC STAINS?

A
  • methylene blue
  • crystal violet
  • safranin

**have to be heat fixed before we apply to make bacteria stick and be killed

29
Q

Describe SIMPLE STAIN PROCEDURE

A
  1. Add small drop of water on slide with loop
  2. Add bacteria into water
  3. Allow smear to air dry
  4. Use slide holder and heat fix
  5. Allow to cool and add stain on top of staining tray
  6. Rinse with water after some time
  7. Blot dry with bibulous paper
30
Q

Describe NEGATIVE STAINS

A

Use of specific DYE SOLUTION that is now ACIDIC IN NATURE
- (has specific hydrogen ion being given up creating a NEGATIVE CHARGE)
CHARGES REPEL EACH OTHER

**CELL IS UNSTAINED

31
Q

Why do we use negative stains?

A

To determine morphology and cellular arrangement and bacteria

  • OFTEN USED IF BACTERIA CANT STAND HEATFIXING 
32
Q

Describe NEGATIVE STAIN PROCEDURE

A

using BLACK STAIN—NIGROSIN

  1. Add drop of black dye on the side of the slide.
  2. using aseptic method, mix organism onto the dye with loop
  3. use a second clean slide and hold it at a 30° angle, then spread it across the slide.&raquo_space;»> then «&laquo_space;lmao

NO HEAT FIXING

33
Q

Describe GRAM STAINS

A

defined as a DIFFERENTIAL STAIN—use of a DECOLORIZATION STEP

  • specific use of a PRIMARY STAIN (CRYSTAL VIOLET)
    + the special addition of IODINE (MORDANT) (greater affinity to cell wall)

CREATION OF THE CRYSTAL VIOLET IODINE COMPLEX

34
Q

 why is depolarization the most crucial step in the gram stain procedure? what OTHER STAIN IS IMPORTANT?

A

The DECOLORIZER REMOVES COLOR FROM GRAM NEGATIVE CELLS ONLY +gram positive keep the dye

Can use a COUNTERSTAIN (SAFRANIN) to color GRAM NEGATIVE CELLS

GRAM - = RED
GRAM + = PURPLE

35
Q

What is the difference between gram-positive and gram-negative cell?

A

GRAM NEGATIVE CELLS
have thinner Peptidoglycan layers
**CANNOT HOLD VIOLET DYE—ALCOHOL IN DECOLORIZER BEGINS TO REMOVE FAT FROM LAYER + CANNOT KEEP COMPLEX INTACT

Gram-positive cells
have sticker Peptidoglycan layers

36
Q

Describe over decolorizing and under decolorizing

A

OVER DECOLORIZE
can get reddish gram-positive cells

UNDER DEPOLARIZE
can get purpleish gram-negative cells

37
Q

describe gram stain procedure

A
  1. Heat fixed emulsions
  2. Cover with CRYSTAL VIOLET STAIN (1 MIN)
  3. RINSE AT ANGLE with WATER
  4. COVER WITH IODINE (1 MIN)
  5. RINSE AT ANGLE with WATER
  6. begin DECOLONIZING—until runoff is clear (ONCE CLEAR—IMMEDIATELY RINSE WITH WATER)
  7. begin COUNTERSTAIN with SAFRANIN (1 MIN)
  8. RINSE AT ANGLE with WATER
  9. blot dry

done :) 😁

38
Q

Describe ACID FAST STAINS + what STAIN are we using?

A

looking for the PRESENCE OF MYCOLIC ACIDS in ACID FAST ORGANISMS

MYCOLIC ACID:
waxy substance that gives better binding to the primary stain and resistance to decolorization

ACID FAST POSITIVE ORGANISMS:
- repels AQUEOUS STAINS
- weakly GRAM POSITIVE

STAIN:
- stain of PHENOLIC COMPOUND CARBOLFUSCHIN
- lipid soluble + penetrates cell wall
- use of COUNTERSTAIN: METHYL BLUE

39
Q

how do acid-fast positive and acid-fast negative cells stain?

A

ACID FAST POSITIVE
stain red-purplish

ACID FAST NEGATIVE
methyl blue

40
Q

ZN method vs K method

A

ZN METHOD
- use of steam heating to get stain of CARBOLFUCHSIN into cell wall by MELTING WAX

K METHOD
- just using STRONGER CONCENTRATIONS OF CARBOLFUCHSIN

41
Q

Describe ACID FAST STAINING PROCEDURE

A
  1. Heat fixed emulsion
  2. Add and apply STAIN CARBOLFUCHSIN for 5-10 MIN
  3. Rinse with distilled water
  4. Begin decolonizing with ACID-ALCOHOL
  5. Rinse with distilled water
  6. Stain now with METHYL BLUE
  7. Rinse with distilled water
  8. Blot dry
42
Q

Describe ENDOSPORE STAINS (more specifically ENDOSPORES

A

Trying to look and differentiate specific DORMANT CELLS OF ENDOSPORES

  • only come out in NUTRIENT DEPLETION or HIGH TEMPS
  • EXTREMELY RESISTANT TO HEAT AND CHEMICALS
  • has outer covering of KERATIN
  • dependent on POPULATION DENSITY—the “competence and sporulation factor” CSF
  • we become VEGETATIVE CELLS once in suitable conditions

MICROBIO BIOL-160 LEC (21 decks)

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