Microbio LAB - 2ND MEETING

AUTOCLAVE

an instrument used for sterilization of culture media, glassware like test tubes, petri dish, and pipettes. most commonly used equipment for sterilization

121°C - 20 minutes - 15lbs

Autoclave

The temperature of this instrument must be set to _____, time of exposure is _____, pressure at _____. produced inside this instrument

AUTOCLAVE

Principle: steam under pressure

LABORATORY OVEN

• This equipment is used for sterilization of glassware and for drying.

• The materials are exposed for 30 minutes at a very high temperature of 150°C

LABORATORY INCUBATOR

an instrument that provides an optimum temperature for growth of microorganism used for cultivation of microorganism inoculated or planted in a culture media

AUTOCLAVE

an instrument used for sterilization of culture media, glassware like test tubes, petri dish, and pipettes. most commonly used equipment for sterilization. The temperature of this instrument must be set to 121°C, time of exposure is 20 minutes, pressure at 15lbs. produced inside this instrument. Principle

LABORATORY OVEN

• This equipment is used for sterilization of glassware and for drying.

• The materials are exposed for 30 minutes at a very high temperature of 150°C.

LABORATORY INCUBATOR

an instrument that provides an optimum temperature for growth of microorganism used for cultivation of microorganism inoculated or planted in a culture media. optimum temperature for growth of bacteria is 35-37°C.

MILLIPORE FILTER

an apparatus used for removing bacteria in liquid and sterilization of culture media and substances that can not tolerate the high temperature of autoclave and oven.

WATER BATH

used for short term incubation or growth of microorganism. the temperature of the water bath for incubation is 37 °C.

PETRI DISH/CULTURE TUBE

these apparatus are used as container for culture media and cultures in order for the microorganism to propagate or grow and multiply.

LABORATORY REFRIGERATOR

an instrument used for storage of culture media, and antibiotic disk.

DURHAM FERMENTATION TUBE

a small test tube used to detect gas production.

CANDLE JAR

an apparatus used for bacteria that require an increase CO2 for their growth. lighted candle jar placed inside and covered with a tight fitting closure. once the cover is fitted into the jar, the flame will disappear leaving a smoke that represents CO2.

CONCAVE SLIDES

a special glass slide used to detect movement or motility of bacteria.

INOCULATING LOOP/NEEDLE

an apparatus used for planting and transferring microorganisms.

ERLENMEYER FLASK WITH BEADS

this apparatus is used for defibrination of blood. pag nagprepare ng CAP, need to remove the fibril present sa blood. isswirl yung bead.

GLASS SLIDES

a piece of glass where the specimen is placed for microscopic examination.

STIRRING ROD

a piece of glass tubing used for mixing.

WASH BOTTLE

an apparatus used to wash out stains from microbial smears.

ALCOHOL LAMP

this apparatus is used to sterilize inoculating loop/needle as well as flaming the sides of petri dish and mouth of test tube to prevent contamination.

PIPETTES

an apparatus used for measuring and transferring fluid.

MORPHOLOGICAL STUDY

can be done whether the organisms are in the living or fixed state.

LIVING STATE

"wet mount" - best observed for the shape & arrangement of organisms.

HANGING DROP

best observed for the motility.

FIXED STATE

adhere the organism on the slide by fixing so that the organisms may not be washed off during staining.

HANGING DROP METHOD

drop is suspended in a concave slide for motility.

SIM (SULFIDE INDOLE MOTILITY MEDIUM)

a semi solid butt, depth is 5cm. stab the inoculum once vertically into the water of the agar, butt approximately 2cm depth. non-motile growing along the stab line, however the growth along the or growth of organisms away from the stab line is a positive modification of motility.

STAINING THE FLAGELLA

by simple stain, Leifson stain.

SEROLOGICAL TEST

flagellar antigen & antibody.

FLUORESCENT ANTIBODY TEST

detects flagella with fluorescent dye.

SWARMING PHENOMENON

spreading growth over agar surface indicating motility.

COCCUS (COCCI)

round, spherical or ellipsoidal in shapes (singly = coccus; in pairs = diplococci; in chain = streptococci; in bunch/group = staphylococci; in group of 4 = tetrads; in group of cuboidal/pockets of 8 = sarcinae).

BACILLUS (BACILLI)

cylindrical or rod-like shape (singly = bacillus; in pairs = diplobacilli; in chain = streptobacilli; Chinese character-shaped organisms tend to bend at point of division resembling a Chinese character).

SPIRILLUM (SPIRILLI)

spiral shape, differs in length, number and amplitudes of spiral

• vibrio

• spirillum

• spirochetes

TERMINAL SPORE

one end (Clostridium tetani).

SUBTERMINAL SPORE

near the end (Clostridium sporogenes).

CENTRAL/EQUATORIAL SPORE

spore in the center.

STAINING

the process of artificially coloring the microorganisms with the use of dyes.

PURPOSE OF STAINING

to differentiate one organisms from one another to observe and appreciate the appearance of microorganisms; helps in the identification of microorganisms and its special structure.

SIMPLE STAINING

• used only one particular dye to stain the organism

• resulting color is the dye color

  • crystal violet = violet

  • safranin = red/pink

DIFFERENTIAL STAINING

used two or more dyes to stain the organisms and differentiate one group from another.

GRAM STAIN

most widely used differential staining method in microbiology. used to differentiate the gram (+) to gram (–) microorganisms.

CRYSTAL VIOLET

primary stain, stains all organisms in the smear.

GRAM’S IODINE

mordant, enhances or strengthens the union between the primary dye and its substrate.

95% ETHYL ALCOHOL

decolorizer, dissolves and removes the primary stain from the cells.

SAFRANIN

counterstain.

GRAM STAIN

Gram (+) = violet or purple; Gram (–) = red or pink.

Except:

• Neisseria

• Veillonella

• Branhamella

All cocci are G (+)

ACID FAST STAIN

differentiates acid fast organisms from non-acid fast organisms, especially for mycobacteria group.

ACID FAST ORGANISMS

very hard to stain but once stained they are very hard to decolorize.

ZIEHL-NEELSEN METHOD

employs steaming process.

KINYOUN’S METHOD

no steaming process, instead of methylene blue uses malachite green.

PAPPENHEIM’S METHOD

used to differentiate M. lacticola or M. smegmatis from M. tuberculosis (decolorizer = rosalic acid).

BAUMGARTEN’S METHOD

used to differentiate M. leprae from M. tuberculosis.

CARBOL FUCHSIN

primary stain in acid fast staining.

ACID ALCOHOL

decolorizer in acid fast staining.

METHYLENE BLUE

counterstain in acid fast staining.

RESULT IN ACID FAST STAIN

acid fast = red; non-acid fast = blue.

RULE IN ACID FAST STAINING

all bacilli are non-acid fast except Mycobacteria group.

CELL WALL STAINING

Dyar method (plant cell only).

SPORE STAINING

Heat & Acetic method, Schaeffer-Fulton spore stain, Dorner’s spore stain.

CAPSULAR STAINING

• Tyler capsule stain

• Hiss capsule stain

• Anthony’s method

• Welch’s capsule stain

FLAGELLAR STAINING

Gray’s method, Loeffler’s method, Leifson method, Van Emergen method, Caesares Gil method.

METACHROMATIC GRANULES

Loeffler’s Alkaline Methylene Blue (LAMB), Neisser method, Alberts method.

INDIRECT/RELIEF OR NEGATIVE STAINING

process in which the background and not the organism are the ones stained, microorganisms appear as colorless objects

• India ink or Barris method

• Nigrosine method

culture media

anything that possess nutritional and environmental requirement for bacterial growth.

CULTURE

a group of microorganisms obtained in a culture medium.

COLONY

a crop or growth of organisms.

PURE CULTURE

culture containing one organism only.

MIX CULTURE

culture containing two or more organisms.

STOCK CULTURE

culture that is readily made.

LIQUID MEDIUM

culture medium in liquid state.

SEMI-SOLID MEDIUM

culture medium with 0.01% agar.

1. Synthetic Medium

2. Non-synthetic Medium

3. Tissue Medium

According to composition

1. Plated Medium

2. Tubed Medium

According to how medium is dispensed

1. Simple Medium

2. Enrichment Medium

3. Enriched Medium

According to use

1. Liquid Medium

2. Semi-solid Medium

3. Solid Medium

According to consistency or physical state

Simple Medium

Supports only the growth or multiplication of microorganisms and also used for routine cultivation.

e.g. NA (nutrient agar)

Enrichment Medium

Enhances the propagation of certain organisms

e.g. SB, TTB

Enriched medium

Contains nutritive supplements needed for the growth of some organisms.

e.g. BAP, CAP

Differential Medium

Distinguishes organisms growing together by differences in their cultural characteristics

e.g. EMB, Mac Conkey

Selective Medium

Promotes the growth of desirable organisms but at the same time inhibiting the growth of others.

e.g. BSA

Special or Specific Medium

Support the growth of specific organisms.

e.g.

• Thayer Martin - Neisseria

• Petragnani - Mycology-fungi

• Mac Bride agar - Listeria

1. Liquid culture medium

2. Slant medium

3. Butt medium

4. Butt/Slant

5. Plated medium

TECHNIQUES OF INOCULATION (depends on the type of culture medium)

• Nutrient broth

• Carbohydrates media

• Nutrient or plain agar blood agar

• Chocolate agar

• Loefflers blood serum

• Endos medium

Culture media that are commonly used in the laboratory:

LIQUID CULTURE MEDIUM

(acc to technique of inoculation) inoculate the medium by shaking.

SLANT MEDIUM

(acc to technique of inoculation) streaking from the bottom with zigzag fashion.

BUTT MEDIUM

(acc to technique of inoculation) by stabbing.

BUTT/SLANT

(acc to technique of inoculation) stabbing in the butt portion and then streaking with a zigzag fashion in the slanted portion of the medium.

PLATED MEDIUM

(acc to technique of inoculation) streaking.

Except:

• Corynebacterium

• Clostridia

• Bacillus

• Listeria

• Erysepelothrix

• Insidiosa.

All cocci are G (-)