PURE CULTURE ISOLAtion

BACTER35 PRENE G. PAREDES, RMT

Objectives:

At the end of the class, the students are expected to:

1.Learn the different concept in pure culture isolation and biochemical testing.

2.Apply these concepts to the next laboratory activities

3.Develop skills in identifying unknown bacteria.

Pure Culture

Most infectious materials contain several kinds of bacteria. If they are plated onto the surface of the solid medium, colonies will form that are exact copies of the same organism.

A visible colony arises from a single spore or vegetative cell or from a group of the same organisms attached to one another in clumps or chains

Microbial colonies often have distinctive appearance that distinguishes one microbe from another .

The bacteria must be distributed widely enough so that the colonies are visibly separated from each other.

Cultural Characterisitcs

• Hemolysis• Size• Form of Margin• Elevation• Density• Color• Consistency• Pigment• Odor

Isolation Technique

• In nature microbial cultures are mixed

• Identification relies upon isolating individual colonies

• Testing requires pure cultures

• As a result isolation technique provides an essential microbiological tool

Can an isolated colony be considered pure?

This is generally assumed, however….

• some colonies are very slow growers and may be too small to see.

• some colonies may be growing under another colony

• selective media may be preventing reproduction of some bacteria so they may be present but not visible

• condensed water, capsules, slime, all represent areas where individual contaminant cells hide out.

Streak Plate Method

• An original inoculum containing a mixture of bacteria is spread into 4 quadrants on solid media.

• The goal is to reduce the number of bacteria in each subsequent quadrant.

• Colonies are masses of offspring from an individual cell therefore streaking attempts to separate individual cells.

• Discrete colonies form as the individual cells are separated and then multiply to form isolated colonies in the later quadrants.

Streak Plate Method

• Isolation method most commonly used to get pure cultures

• Isolated colonies can be picked up by an inoculating loop and transferred to a nutrient medium to form pure culture

• Works well when the organism to be isolated is present

in large numbers relative to the population

• Useful for studying morphology and hemolytic properties

• Loop is generally used when inoculating specimen

• Needle - When transferring pure cultures from plates to tubes

• Swab Specimen- roll the cotton swab over a small area of the surface at the edge, then streak out from this inoculated area

 

• Plunging a hot wire loop into the specimen should be discourage because this may cause:

• Contamination of the environment by bacterial aerosol

• Death of microorganism in the specimen

• Bacterial aerosol- rough agar surfaces; excessive vibration or rattling of the wire loop within the tube or the plate

Patterns of Streaking and its Purpose 

Experience may dictate the method

of choice

Colony Description

Hemolysis

Special Inoculation Method

• Pour Plate Method used to estimate microbial population

-used to study special characterisitcs of pure cultures such as hemolytic pattern

Qualitative Determination of Bacterial Population

• 1. Standard/ Viable Count

• 2. Spectophotometric (Turbidimetric) Analysis

 

Standard Plate Count Method

• is an inidirect measurement of cell density• -information on live bacteria related• -dilution using sterile saline or phosphate buffer

diluent• - 25-250 colonies in the final plate• - < 25TFTC- unacceptable for statistical reasons• > 250 colonies TMTC- produce colony close to

each other to be distinguished as CFU

• Greater Accuracy is achieved by plating duplicates or triplicates of each dilution

• Dilution Blanks: Sterile Buffered Water

0.85 % NaCl

sterile culture medium

Spectrophotometric Analysis

• is based on Turbidimetry and indirectly measures all bacteria dead or alive

• Turbidity- ability of particles in suspension to refract and deflect light rays passing through the suspension such that the light is reflected back into the eyes of the observer

Optical Density - measurement of turbidity•determined in a spectrophotometer•the amount of light scattered depends on the number and size of the particles in suspension•the amount of light transmitted decreases as the cell population increases

Quantitative Methods of Culturing Urine Specimen

• The recovery of microorganisms in the urine sample does not categorically indicate a urinary tract infection, because even urine sample collected by catheter may become contaminated.

• Liquid soap containing hexachlorophene- suitable cleansing agent

• Specimen from patients with the UTI contains significantly high numbers of bacteria

Quantitation Methods 

• 1. Calibrated loop, Direct Streak Method or Platinum Loop technique

- traditional method in which a known amount of uncentrifuged urine is measured with either 0.01 or 0.001 mL loop and placed on two different media 

• Bacteria/ mL= # colonies/ quantity of urine 

2. Plate Count

3. Turbidimetric Method 

Pour Plate

• used when specimen contain unusually high and mixed population as to make differentiation of species difficult or impossible

• Double Poured Plate

-suitable for large numbers of specimen

Catalase test

It converts 3% H2O2 to oxygen and water, resulting in immediate bubling. Staphylococci are positive.

Streptococci and enterococci are negative.

Catalase test

• Catalase is an enzyme that decomposes hydrogen peroxide into oxygen and water.  Excluding the Streptococci, most aerobic and facultative anaerobic bacteria possess catalytic activity.

• Hydrogen peroxide forms as one of the oxidative end products of aerobic carbohydrate metabolism.  Catalase converts hydrogen peroxide into water and oxygen.  The catalase test is commonly used to differentiate streptococci (negative) for staphylococci (positive).

Coagulase Test

Principle: This test is used to differentiate S. aureus( Positive) from coagualse negative staphylococci(negative)

-S. aureus produces two forms of coagulase: bound and free.

- Bound Coagulase, or “Clumping Factor”,is bound to the bacterial cell wall and reacts directly to fibrinogen

-This rersults in an alteration of fibrinogen so that that it precipiatates on the staphylococcal cells, causing the cells to clump when a bacterial suspension is mixed with plasma

Coagulase Test

• Free Coagulase- an extracellular protein enzyme that causes the formation of

clot when S. aureus colonies are incubated with plasma.

• - the clotting mechanism involves the activation of a plasma COAGULASE REACTING FACTOR(CRF) which is modified or dervied thrombin molecule, to form a coahulase- CRF complex. This complex in turn reacts with fibrinogen to produce the fibrin clot

Coagulase Test

• A. Slide Coagulase

Detects: Cell bound coagulase

Reagent: Rabbit’s Plasma

Positive: formation of clot; if negative confirm with tube coagulase

Coagulase Test

• B. Tube Coagulase

Detects: Free Coagulase

Reagent; 0.5 mL Rabbit’s plasma + colony incubated 37 c

Positive: gel like clot

Coagulase Test

NOTES:

• Check every 30 minutes for 4 hours to avoid false (-) because of Staphylokinase

• If negative after 4 hours confirm with 20 hours incubation

• False positive coagulase test: citrate anticoagulant because citrate utilizing organism release calcium

Manitol salt agar

fermentation results in a color change from pink to yellow. Salt concentration of 7.5% inhibits most organisms other than staph.

All staph can grow on MSA. Only S. aureus, and some S. saprophyticus, cause color change.

Novobiocin

• Organisms that are resistant grow to the edge of the disk. S. Saprophyticus is resistant. Other coag-neg staph are susceptible. Test is performed on coag-neg staph from urine.S. epidermidis is sensitive

Bacitracin disk

• Group A strep is susceptible. Also known as the A disk. No longer recommended because some groups C and G are also susceptible.

SXT

• Group A and B strep are resistant. Group C and G strep are susceptible. Used in combination with bacitracin disk to differentiate group A strep from group C or G strep.

PYR

• If PYR is hydrolyzed, a red color develops after addition of a color developer. Group A strep and enterococci are positive. More specific than bacitracin for group A strep. Group A strep is the only species of beta-hemolytic strep that is PYR-positive

CAMP test

• Group B strep enhances hemolysis of beta-hemolytic S. aureus. An arrowhead formation is seen at the juncture of the two organisms.

Hippurate hydrolysis

If hippurate is hydrolyzed, a precipitate forms when ferric chloride is added to the medium. Group B strep is positive.

Optochin disk

• S. pneumoniae is susceptible. Also known as P disk

Lysine Iron Agar

• This test is used to determine whether an organism decarboxylases or deaminates lysine and forms hydrogen sulfide(H2s)

• Contains lysine(amino acid), glucose( carbohydrate source), protein, bromcresol purple(indicator) and sodium thiosulfate/ ferric ammonium citrate(sulfur source and hydrogen sulfide indicator)

• Acid- Yellow/red

• ‘Alkaline- Purple

• Butt- Lysine Decarboxylation

• Slant- Lysine Deamination

Lysine Iron Agar

Lysine Iron Agar

• Alkaline (purple) slant and Alkaline (purple) butt (K/K)- organism decarboxylate lysine and cannot deaminate it.

- initially the organism ferments glucose, causing production of acid and changing the indicator in the butt to yellow

- the organism then decarboxylates lysine to produce cadaverine(alkaline product) which cause the pH indicator to change from yellow back to purple

Lysine Iron Agar

• Alkaline (purple) slant and Acid( Yellow) butt (K/A)

- organism fermented the glucose but was unable to deaminate or decarboxylate the lysine

Lysine Iron Agar

• Bordeaux Red Slant and Acid(yellow) butt(R/A)

-organism deaminated lysine but could not decarboxylate it. The yellow butt is caused by glucose fermentation. The red slants resuluts from the product of lysine deamination combining with ferric ammonium citrate and flavin mononucleotide to form a burgubdy color on the slant

Lysine Iron Agar

Simmon Citrate Agar

• Useful in differentiating gram negative enteric bacilli in its ability to use citrate as its sole source of carbon.

• Citrate utilizers use the medium ammonium salt as nitrogen salt

• At alkaline pH, the incorporated pH indicator bromthymol blue shifts from green to blue( positive)

Methyl Red Voges- Proskauer Medium

• Useful in distinguishing members of Enterobacteriacae

• Methyl Red

- organism that produce enough acid will overcome the neutralizing effect of the buffer.POSITIVE RESULT:RED

Methyl Red Voges- Proskauer Medium

• Voges Proskauer

• Detects; Acetoin

• Reagents: Acetoin+ KOH - diacetyl + alpha napthol

• Positive result: pink- red color

Triple Sugar Iron

• Used to determine whether a gram negative rod is a glucose fermenter, or non glucose fermenter; sucrose fermenter and hydrogen sulfide producer

• TSI agar contain 10 parts lactose, 10 parts sucrose and 1 parts glucose

• Phenol red(indicator); peptone( carbon, nitrogen source); and sodium thiosulfate plus ferric ammonium sulfate (sulfur source and hydrogen sulfie indicator)

Triple Sugar Iron

• Acid – yellow

• Alkaline- red

• A/AG h2s- lactose, sucrose glucose fermenter

• K/A h2s- Non lactose, Non sucrose, glucose fermenter

• K/K h2s- non LF, non SF, non GF

Triple Sugar Iron

Sulfide Indole Motility

• Indole is a by-product of the metabolic breakdown of the amino acid tryptophan used by some microorganims.

• The presence of indole in a culture grown in a medium containing tryptophan can be readily demonstrated by adding Kovac’s reagent to the culture.

• If indole is present, it combines with the reagent to produce a brilliant red color.

Sulfide Indole Motility

• If it is not present,there will be no color except that of the reagent itself. This test is of great value in the battery used to identify enteric bacteria

• Hydrogen sulfide is produced when amino acids containing sulfur are metabolized by microorganisms.

• If the medium contains metallic ions, such as lead, bismuth , or iron (in addition to an apporpriate amino acid), the hydrogen sulfide formed during growth combines with the metallic ions to form metal sulfide that blackens the medium)

Urease

• Urease broth is a differential medium that tests the ability of an organism to produce an exoenzyme, called urease, that hydrolyzes urea to ammonia and carbon dioxide. 

• The broth contains two pH buffers, urea, a very small amount of nutrients for the bacteria, and the pH indicator phenol red. 

• Phenol red turns yellow in an acidic environment and fuchsia in an alkaline environment. 

• If the urea in the broth is degraded and ammonia is produced, an alkaline environment is created, and the media turns pink.

Bile Esculin Test

• PURPOSE To isolate and identify bacteria able to hydrolyze esculin in the presence of bile.

• Commonly used for presumptive identification of group D streptococci and enterococci, all of which are positive.

• Group D streptococci and enterococci include opportunistic pathogens such as Enterococcus faecalis, Enterococcus faecium, and Streptococcus bovis.

•PRINCIPLE: Bile esculin medium contains esculin and peptone for nutrition and bile to inhibit Gram-positive bacteria other than Group D streptococci and enterococci. •Bile esculin azide agar uses sodium azide to inhibit Gram-negative bacteria.•Ferric citrate is added as a color indicator.

• Esculin is a glycoside (a sugar molecule bonded by an acetyl linkage to an alcohol) composed of glucose and esculetin.

• These linkages are easily hydrolyzed under acidic conditions.

• Many bacteria can hydrolyze esculin, but few can do so in the presence of bile.

ESCULINACID

b-D-Glucose + ESCULITIN

GLYCOLYSIS

Fe+3 Dark Brown Color

Selected Bacteriologic Culture Media

page 976 in your Bacteriology Book