Activity 8-12 Flashcards
-polysaccharide derived from red algae is added to solidify nutrient liquid media
-allowed specimen to be spread across the surface of the medium and grown to produce single colonies, isolating the various microorganisms which are present
-suited as a solidifying agent because it will not melt until the medium is heated to near boiling(95degree C- 100 degree C) but will remain melted untilcooled (42 degree C)- agar solidifies at this temperature
-allow strict control of the growth factors
Agar
Culture media are sterilized by?
Autoclaving
Media that contains heat labile components like G, A, U, S, B ARE NOT AUTOCLAVE. What are they?
glucose, antibiotics, urea, serum, blood
Once the media is prepared, it is held at ________ degrees in the refrigerator for ________ weeks. How many degrees and weeks?
4-5°C;1-2weeks
For Blood Agar, cool melted agar base to about 45-50°C before adding whole blood (5ml/100ml of blood agar base)
Fact
Mix blood with agar by rotating media and making figures of 8, w/o producing bubbles
Fact
For Chocolate Agar, cool agar base to about 50- 60 degreeC before adding whole blood.
Fact
What will lyse RBC giving the medium a chocolate brown color?
High Temperature
the lysis releases intracellular nutrients such as ________ and ____________?
hemin (X Factor); coenzyme Nicotinamide Adenine Dinucleotide (NAD or V Factor)
What will inactivates the enzymes NADases that hydrolyze “V Factor”
Heat
CONTROL of microorganisms is essential in order to prevent the transmission of diseases and infection, stop decomposition and spoilage, and PREVENT UNWANTED MICROBIAL CONTAMINATION microorganisms are controlled by means of physical and chemical agents
Autoclave
Physical Agents?
High or low temperature, Dessication, Osmotic Pressure, Radiation, Filtration
chemical agents?
disinfectants, atibiotics, chemoterapeutic antimicrobial chemicals
Fact
Microorganisms have a minimum, an
optimum, and a maximum temperature for growth
Temperatures below the minimum usually have ______ action on microorganisms
Static
they inhibit microbial growth by slowing down metabolism but do not necessarily kill the organism
Static Action
Temperatures above the maximum usually have __________ since they denature microbial enzymes and other proteins.
Cidal action
is a very common and effective way of controlling microorganisms
Temperature
vegetative microorganisms can generally be killed at _________ with moist heat.
50-70°C
Fact
Bacterial endospores are very resistant to heat and extended exposure to much higher temperature is necessary for their destruction
high temperature may be applied as either moist heat or dry heat
is generally more effective than dry heat because of its ability to penetrate microbial cells
kills organism by denaturing their
protein (causes proteins and enzymes to lose their three dimensional functional shape).
it also may help lipids in cytoplasmic membranes
Moist Heat
Autoclaving employs?
Steam under pressure
During autoclaving, the materials to be sterilized are placed under ___ pounds per square inch of pressure in a pressure cooker type of apparatus.
15; raised to 121°C; 15 minutes
temperature to sufficiently kill bacterial endospores
121°C
is cidal for both vegetative organisms and endospores
is the most common method of sterilization for materials not damaged by heat.
Autoclaving
(Parts of the pressure cooker)
holds the chamber
Body
(Parts of the pressure cooker)
where apparatus or media to be sterilized is places
Chamber
measure the pressure in the chamber
Pressure Gauge
regulates the pressure inside the chamber
Pressure Relief Valve
an exhaust for steam when pressure is reduced
Air Evacuation Valve
gives simultaneous readings for pressure and temperature
Pressure Temperature Thermometer
fasten the lid orcover to the body
Wing nut compression screews
the streak plate technique is used to spread
million of cells over the surface of a solid medium so that some individual cells are deposited at distance from all others.
- these cells grow and reproduce, forming an isolated colony.
Isolation Techniques
Example of streaking method of isolation:
- Four way streak method
- Urine culture streak method
- Three way streak method
- S- Streak method
For incubation, what are the degrees for it and until what time?
place the plate in 35-37 degree celcius incubator for 18-24 hrs
T Streak Method
- draw T on the bottom of petri dish
- aseptic technique is observed
- the loop should be parallel to agar surface to prevent digging into the agar
- streak about half of agar plate
-make one streak into the inoculated portion of the plate. finish b streaking about 1/4 of uninoculated plate.
Pour Plate Technique
One in which a small amount of inoculum from broth cultures/liquid specimen is added by pipette into a molten, cooled agar medium (45-50 degree C)
- pour plates allow microorganisms to grow broth on the surface within the medium.
- most of the colonies grow within the medium and are small in size; few that grow on the surface are oyn the same size and appearance as those on a streak plate.
tube media maybe in form of _______,_____,______ depending on the purpose of the inoculation and the type of medium used, inoculating loop or wire loop may be used.
solid agar slants, semi solids or broths,
place the loop at the base of the slant surface and draw it up the agar surface while moving it from side to side.
Agar slants
insert the inoculating needle and stab it in the center of the agar tube to the bottom. draw the needle out carefully, keeping it straight.
Agar butts
Used to test large volume of liquid samples, including water and filterable beverages
Membrane Filtration Method
used as enrichments and general cultivation
Innoculation of Broth Media
contains two or more bacterial species that are known and can be easily separated based on cultural or biochemical characteristics.
Mixed Culture
provides a means for
maintaining adequate nutrition for the organisms so they can continue to survive
Culturing Techniques
Culturing procedures
Isolation techniques
is a colony that is not touching any other colonies and assumed to be a pure culture. these colonies are easily accessible for performing staining identification processes.
Isolated Colony
is a very homogeneous population formed by the progeny of a single bacterium
Bacterial Colony
Seven macroscopic characteristics of bacterial colonies
Size, Shape(Form), Margin, Surface Texture, Pigmentation, Transmitted light/ optical property, & Elevation
whenever possible measure colony diameter and express in mm
Size
Size
- punctiform- to small to measure
- moderate- 1mm-5mm
- large- 5-10mm
Shape/Form
Circular, Irregular, Filamentous, Rhizoid
outer edge/periphery of the coloy
Margin
Margin
- entire- even
- Undulate/wavy
- Filamentous
- Erose/serrated
- Lobate/Curled
Surface Texture
- Rough/coarse
- smooth/shiny
- mucoid
- wrinkled
the color as precisely as possible
Pigmentation
Transmitted light/ optical property:
Transparent, Opaque, Transluscent
the light passes to the colony; clear
Transparent
if the light does not pass through
the colony
Opaque
if the light can pass in a diffuse manner so that a colony is illuminated but objects at the other side cannot be seen directly
Translucent
Elevation
- flat raised
- convex/dome shape
- umbonate
enriched, general supportive and
differential medium used to grow a variety of fastidious microorganism such as Streptococcus.
- can function as differential media because it can demonstrate different types of hemolysis such as:
Beta, Gamma(Non), Alpha hemolysis
Blood Agar
complete lysis of RBC by streptolysin O and streptolysin S enzymes; which produces clear halo around the colonies
Beta hemolysis
incomplete lysis of rbc resulting in breakdown of hemoglobin, which produces greenish halo around bacterial colonies
Alpha Hemolysis
no lysis of rbc; no
significant change in color of the agar surrounding the colonies
Gamma (Non) Hemolysis
provide serum ingredients and
erythrocytes which would allow differentation of microorganisms growing based on their
hemolytic reactions.
Blood
-selective media
-grows only staphylococcus bacteria
-7.5% salt is inhibitory to most other bacteria
- it is a nutritive medium due to its contents of peptones and beef extract, which supply essential growth factors such as nitrogen, carbon, sulfur and trace nutrients
- also a differential media used to distinguish between Staphylococcus aureus and other Staphylococcus spp.
Mannitol Salt Agar (MSA)
Fact
mannitol fermenation with subsequent acid production by S. aureus is indicated by a change in the color of the phenol red indicator to yellow
the name of the enzyme found in
most bacteria which initiates the breakdown of hydrogen peroxide (H202) into water (H20) and free oxygen (02)
Catalase
Fact
Most bacteria are catalase (+)
Catalase (-)
certain genera that dont carry out aerobic respiration, such as
Streptococcus, Lactobacillus, and Clostridium
The catalase test differentiates the
Staphylococci
Streptococci
Streptococci catalase ?
-
Staphylococci catalase ?
+
Oxygen bubbling in catalase test?
Positive
Little to no bubbling in the catalase test?
Negative
Little to No bubbling in catalase test
Negative
Little to No bubbling in catalase test
Negative
Fact
Do not pick colonies from BAP for catalase test because RBC contains catalase. This may lead to false positive reaction.
- used to differentiate Staph aureus (coagulase positive) from coagulase negative staphylococci.
- S. aureus produces two forms of coagulase:
•bound coagulase
•free coagulase
Coagulase Test
is bound to the bacterial cell wall and directly reacts with fibrinogen.
Bound coagulase or clumping factor
macrocopic clumping in 10-15 secs
Positive
No clumping
Negative
