BACTERIAL ISOLATION

Isolation of the microorganisms in microbiology

- process of developing a pure culture by separating one species of microbe from a mixture of many other species.

Pure cultures

biochemical

morphological

cultural characteristics

_________ contain only one type of microorganism and are ideal for studying their _______, ________, and _____________

Subculture

pure culture

mixed culture

Method of Bacterial Isolation

Subculture

Involves transferring cells from an existing culture to fresh growth medium in order to prolong the life and expand the number of cells.

subculturing techniques

Microbiologists use _______________ to grow and maintain bacterial cultures, to examine cultures for purity or morphology, or to determine the number of viable organisms.

culture

A population of bacteria grown in the laboratory is referred to as a ________

pure culture

mixed culture

A __________ contains only one single type of bacteriaa;

● A ________ contains two or more different bacteria.

INOCULATION

INOCULUM

INOCULATION TECHNIQUES

INOCULATION

– the process of introducing microorganisms into a culture medium for cultivation and growth

INOCULUM

– the substance containing bacteria that is used for culture.

a. Inoculating loop

needles (hold like a pencil);

Commonly used materials: FOR INOCULATION

qualitative indications

quantitative depending

Culture colonies are reported via ____________ or ____________ on the sample type

blood

sputum

stool

body fluids

wound

(urine)

–Few, moderate, heavy growth

– for quantitation _________

1. Streak Plate

2. Spread Plate

3. Pour Plate

Techniques to Obtain Pure Culture

ISOLATION STREAK METHOD

2 Different Techniques:

ISOLATION STREAK METHODS

SEMIQUANTITATIVE isolation technique

slight growth

moderate growth

heavy growth

(most common as a MT)• - Also called ______________ because the

• grading for growth of this technique will be depending on which set of streak the growth is observed (1+, 2+, 3+...) (_________, _________, __________)

microbiological laboratory technique

pure cultures

mixed population.

What is Streak Plate Method?

• The streak plate method is a ____________________ of isolating _________, and/or getting well-isolated colonies of bacteria from a ___________

STREAK METHOD

It is mostly used to get pure cultures of bacteria; however, yeasts can also be isolated by this method.

streaking method

It is one of the most commonly used aseptic techniques in microbiology to isolate and propagate bacteria. It is a mechanical isolation technique used in microbiology, commonly known as the “______________”

1. Quadrant Streaking

2. T-Streaking

3. Continuous Streaking

4. Radiant Streaking

5. Semi-quantitative Streaking

6. Zigzag Streaking

Types of Streak Plate Method–

18 to 24

INITIAL OBSERVATION OF CULTURE

Observation of colony morphology is done after __________ hours incubation

Culture medium

Environmental conditions - oxygen carbon dioxide, temperature, pH, and moisture

• Factors affecting growth & colony morphology:

PLATE READING

– Comparative examination of colony morphology of microorganisms growing on various culture media.

Chocolate agar:

most organisms andfastidious organisms

Blood agar

: grows most organisms

Mac Conkey agar

: gramnegative organisms

(pink)

(colorless)

Differentiation using plated media

– Lactose fermenters ______ / nonlactose fermenters _______

PINK

COLORLESS

LACTOSE-FERMENTING GRAM NEG COLOR _______ IN MACCONKEY

NON-LACTOSE FERMENTING GRAM NEG COLOR _______ IN MACCONKEY

ESCHERICHIA/CITROBACTER (DRY)

KLEBSIELLA/ENTEROBACTER (CREAMY) (NOTICE IN 48HRS)

EXAMPLE OF LACTOSE FERMENTING

β hemolysis:

α hemolysis:

TYPES OF HEMOLYSIS:

βhemolysis

: complete clearingof blood cells around thecolonies

α hemolysis

: partial clearing of blood that results in a green discoloration of the medium

Y hemolysis

• Nonhemolytic colonies are referred to as _________.

STREPTOCOCCCUS PYOGENES

STREPTOCOCCUS AGALACTIAE

EXAMPLE OF A B HEMOLYSIS

IN CHOCOLATE AGAR

BECAUSE THE RED CELL IN THE MEDIUM HAVE ALREADY BEEN LYSED (GREEN IN AROUND THE COLONY OF CHOC)

A HEMOLYSIS

SIZE

FORM OR MARGIN

ELEVATION

DENSITY

CONSISTENCY

COLOR

PIGMENT

ODOR

COLONY CHARACTERISTICS

PINPOINT/PUNCTIFORM

SMALL

MEDIUM

LARGE

SIZE:

<1 MM

1-2MM

3-4MM

>5MM

SIZE

PINPOINT/PUNCTIFORM-

SMALL-

MEDIUM-

LARGE-

smooth

filamentous

rough

irregular

FORM OR MARGIN-

Bacillus anthracis is filamentous

Diphtheroid colonies have rough edges

EXAMPLE OF FORM OF MARGIN

raised

convex

flat

umbilicate

umbonate

ELEVATION-

S. Pneumoniae

diphtheroids

Umbilicate: convex with depressed center (pitting); ________________ (if no capsule

– Umbonate: convex with protruding nipple; _________

translucent

transparent

opaque

DENSITY-

b-hemolytic streptococci

example of tranlucent or tranparent

staphylococcus aureus

example of opaque

white

gray

yellow

buff

COLOR- , , or

coagulase negative straphylococci

example of white

nonpathogenic neisseria spp.

yellow

butyrous

creamy

brittle

mucoid

CONSISTENCY-

Neisseria

Nocardia

Streptococci

Diphtheroid

•_______: sticky

• _______: brittle, crumbly, and wrinkled

• __________: dry

• ________: dry and waxy

brick red

PIGMENT

Serratia marcescens:

green, sometimes a metallic sheen

Pseudomonas aeruginosa: ________________

blue

Kluyvera spp:

purple

Chromobacterium violaceum:

brownblack pigment

Prevotella melaninogenica:

old sock

ODOR

S. aureus:

fruity or grapelike

P. aeruginosa:

putrid

• Proteus mirabilis:

musty basement

• Haemophilus spp.:

freshly plowed field

Nocardia spp.:

CRATERIFORM

NAPIFORM

INFUNDIBULAR

SACCATE

STRATIFORM

Describing the Growth of Microbes in Media - Nutrient Gelatin

1.Crateriform

: Liquefied surface area issaucer-shaped.

Napiform

2. : Bulbous-shaped liquefaction at surface.

Infundibuliform

3. : Funnel-shaped.

Saccate

4. : Elongated, tubular.

Stratiform

5. : Complete liquefaction ofthe upper half of the medium.

TURBIDITY

Overall cloudiness of liquid

ESCHERICHIA COLI IN EMB

“greenish metallic sheen

ENCAPSULATED

KLEBSIELLA PNEUMONIAE

“mucoid colonies”