Lab Practical Review Flashcards
definition of a BACTERIAL COLONY
- where bacteria begins to grow on a SOLID SURFACE
- COLONY: a VISIBLE MASS of BACTERIA–arising from SINGLE BACTERIAL CELL
what characteristics are in COLONY MORPHOLOGY (7)?
- 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.)
what other FACTORS make bacteria grow?
- temperature
- time of growth
- nutrient avalibility
definition of MICROSCOPY
the field of USING MICROSCOPES to look at OBJECTS that cannot be seen with the NAKED EYE
what are the THREE WELL-KNOWN BRANCHES of MICROSCOPY?
- LIGHT
- ELECTRON
- SCANNING PROBE
definition of COMPOUND LIGHT MICROSCOPE
- has a SERIES OF LENSES
- “COMPOUND”–magnification of various LENS–giving viewer an ENLARGED INVERTED IMAGE of the object
what are the OBJECTIVE and TOTAL MAGNIFICATIONS?
LOW POWER SCANNING:
4x - 40x
LOW POWER:
10x - 100x
HIGH-DRY:
40x - 400x
OIL:
100x - 1000x
condenser
condenses the light beams on the actual specimen
objective
where the light bend/refracts and creates magnified initial image
ocular lenses
magnifies the final image as virtual image
phase contrast microscope
use of a SPECIAL CONDENSER that BENDS LIGHT
- gives great CONTRAST of INTERNAL STRUCTURES
dark-field microscopes
use of a DARK-FIELD CONDENSER to BLOCK LIGHT from the OBJECTIVE LENSES–appears as LIGHT against DARK BACKGROUND
how do we STORE our MICROSCOPES?
- 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
what is important about EFFECTIVE HAND WASHING?
- helps to MINIMIZE DIRECT PERSON to PERSON + INDIRECT CONTACT transmission of PATHOGENS
how does SOAP play a role in terms of handwashing?
has specific ACTIVE INGREDIENTS to either KILL (BACTERICIDAL) or STOP BACTERIA GROWTH (BACTERIOSTATIC)
definition of CULTURE and PURE CULTURE
CULTURE:
media that contains living microbes
PURE CULTURE:
contains only one species
definition of ASEPTIC METHOD
the transferring of MICROBES from pure culture to a STERILE CULTURE
- avoiding any chance of CONTAMINATION to urself, others, and environment
What are the types of media that we have?
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
What are our transfer instruments?
Inoculating loops, or inoculating needles
Describe procedure of ASEPTIC TRANSFER: BROTH PURE CULTURE to STERILE BROTH TUBE
- Label sterile tube with necessary details
- Use inoculating loop
- Flame and sterilize inoculating loop
- Flame and sterile broth tube
- Remove cap and insert loop—should have a film now
- Reflame tube and close
- Pick up sterile broth tube
- Flame sterile broth tube
- Remove cap
- Mix the INOCULUM into sterile medium
- Remove loop and reflame sterile broth tube (close)
- Flame loop
DONE :)
Describe ASEPTIC TRANSFER: AGAR SLANT CULTURE to STERILE AGAR SLANT
*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 😨
Definition of MIXED CULTURE
microbial culture that contains TWO OR MORE SPECIES
Why do we use the ISOLATION TECHNIQUE on the STREAK PLATE?
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
Quadrant streak method
quadrant streaks—sterilizing loop after first initial streak in quadrant to decrease cell density over time
What is the SPREAD PLATE TECHNIQUE?
A method of ISOLATION —using a DILUTED MICROBIAL SAMPLE onto AGAR PLATE, spread with a glass rod
- can help grow into INDIVIDUAL COLONIES
Describe SIMPLE STAINS—what are CHROMOGENS and CHROMOPHORES?
STAIN:
has a solvent and specific COLORED MOLECULE (CHROMOGEN)
**gives color through CHROMOPHORES (has an AUXOCHROME which is CHARGED)
How do AUXOCHROME AND BASIC STAINS INTERACT?
AUXOCHROME - positively charged
BASIC STAIN - negatively charged
are ATTRACTED TO EACH OTHER—why we have dyed stains
What are our TYPICAL BASIC STAINS?
- methylene blue
- crystal violet
- safranin
**have to be heat fixed before we apply to make bacteria stick and be killed
Describe SIMPLE STAIN PROCEDURE
- Add small drop of water on slide with loop
- Add bacteria into water
- Allow smear to air dry
- Use slide holder and heat fix
- Allow to cool and add stain on top of staining tray
- Rinse with water after some time
- Blot dry with bibulous paper
Describe NEGATIVE STAINS
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
Why do we use negative stains?
To determine morphology and cellular arrangement and bacteria
- OFTEN USED IF BACTERIA CANT STAND HEATFIXING 
Describe NEGATIVE STAIN PROCEDURE
using BLACK STAIN—NIGROSIN
- Add drop of black dye on the side of the slide.
- using aseptic method, mix organism onto the dye with loop
- use a second clean slide and hold it at a 30° angle, then spread it across the slide.»_space;»> then ««_space;lmao
NO HEAT FIXING
Describe GRAM STAINS
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
 why is depolarization the most crucial step in the gram stain procedure? what OTHER STAIN IS IMPORTANT?
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
What is the difference between gram-positive and gram-negative cell?
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
Describe over decolorizing and under decolorizing
OVER DECOLORIZE
can get reddish gram-positive cells
UNDER DEPOLARIZE
can get purpleish gram-negative cells
describe gram stain procedure
- Heat fixed emulsions
- Cover with CRYSTAL VIOLET STAIN (1 MIN)
- RINSE AT ANGLE with WATER
- COVER WITH IODINE (1 MIN)
- RINSE AT ANGLE with WATER
- begin DECOLONIZING—until runoff is clear (ONCE CLEAR—IMMEDIATELY RINSE WITH WATER)
- begin COUNTERSTAIN with SAFRANIN (1 MIN)
- RINSE AT ANGLE with WATER
- blot dry
done :) 😁
Describe ACID FAST STAINS + what STAIN are we using?
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
how do acid-fast positive and acid-fast negative cells stain?
ACID FAST POSITIVE
stain red-purplish
ACID FAST NEGATIVE
methyl blue
ZN method vs K method
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
Describe ACID FAST STAINING PROCEDURE
- Heat fixed emulsion
- Add and apply STAIN CARBOLFUCHSIN for 5-10 MIN
- Rinse with distilled water
- Begin decolonizing with ACID-ALCOHOL
- Rinse with distilled water
- Stain now with METHYL BLUE
- Rinse with distilled water
- Blot dry
Describe ENDOSPORE STAINS (more specifically ENDOSPORES
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
