Bls 206 lecture 3

Isolation Technique

In nature microbial cultures are mixedIn nature microbial cultures are mixed

Identification relies upon isolating individual Identification relies upon isolating individual coloniescolonies

Testing requires pure culturesTesting requires pure cultures

As a result isolation technique provides an As a result isolation technique provides an essential microbiological toolessential microbiological tool

Mixed Culture from Raw Poultry

Streak Plate Isolation Principle

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

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

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

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

The Goal -Isolated Colonies to Start Pure Cultures

Can an isolated colony be considered pure?

This is generally assumed, however….This is generally assumed, however….some colonies are very slow growers and may some colonies are very slow growers and may

be too small to see.be too small to see.some colonies may be growing under another some colonies may be growing under another

colonycolonyselective media may be preventing reproduction selective media may be preventing reproduction

of some bacteria so they may be present but not of some bacteria so they may be present but not visiblevisible

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

Any special considerations?

Different species of microbes represent Different species of microbes represent challenges….challenges….

Encapsulated bacteria are sticky and don’t Encapsulated bacteria are sticky and don’t separate well.separate well.

Some species are motile and do not stay where Some species are motile and do not stay where you streak them spreading across the plate.you streak them spreading across the plate.

Fungal spores easily contaminate cultures within Fungal spores easily contaminate cultures within a plate.a plate.

Organisms can gain entrance to a Petri dish Organisms can gain entrance to a Petri dish through water or the edges, or from the air through water or the edges, or from the air currents while you are streaking.currents while you are streaking.

Microbes will surprise you each chance they get !

Isolation Requires Aseptic Technique

Isolation Requires Aseptic Technique

Aseptic technique is the process of:

• Preventing contamination of a culture with environmental microbes

• Preventing contamination of yourself or the environment with the organism in the culture

•Remember everything is contaminated with a variety of environmental microbes.

•Remember microbes are invisible, you must “see with your minds eye” during these procedures.

Streaking the Quadrants

Flame between each quadrant.QQQuuuaaadddrrraaannnttt 111

QQQ 444 QQQ 222

QQQ 333

Quadrant 1- Streak with broad narrow strokes in the upper half of the first quarter of the plate.

Incinerate and cool the loop between the quadrants

Quadrant 2 – Rotate the plate, enter the previous streak mark one or two times and then streak the upper portion of the second quarter of the plate with broad strokes.

Incinerate and cool the loop between the quadrants

Quadrant 3 – Rotate the plate, enter quadrant 2 one or two times and then streak with shorter more separated strokes from the top of the quadrant to the center.

Incinerate and cool the loop between the quadrants

Quadrant 4 – Enter quadrant 3 and then streak with broad S-shaped motions through the center of the plate.

Streaking the Quadrants

Flame between each quadrant.QQQuuuaaadddrrraaannnttt 111

QQQ 444 QQQ 222

QQQ 333

Isolated Colonies

Culturing and Isolation TechniquesCulturing and Isolation Techniques

Bacteria require a constant nutrient supply to survive Bacteria require a constant nutrient supply to survive and growand grow

Acquire nutrients from their surroundings (free-living) Acquire nutrients from their surroundings (free-living) or from a host (parasites)or from a host (parasites)

Artificial media is used to grow bacteria in a lab (in Artificial media is used to grow bacteria in a lab (in vitro)vitro) Agar is extracted from marine algaeAgar is extracted from marine algae

A carbohydrate that cross-links to form a semi-A carbohydrate that cross-links to form a semi-solid mesh.solid mesh.

Melts at 100 Melts at 100 C, solidifies at 42 C, solidifies at 42 C, but will C, but will remain a liquid at 60 remain a liquid at 60 C.C.

Most pathogenic bacteria have an optimum growth Most pathogenic bacteria have an optimum growth temperature of 37 °C (human body temp.)temperature of 37 °C (human body temp.)

Organisms grown in broth cultures are apparent through Organisms grown in broth cultures are apparent through the turbidity that the large numbers of cells produce in the the turbidity that the large numbers of cells produce in the broth.broth.

On agar, a solid medium, the bacterial cells form masses On agar, a solid medium, the bacterial cells form masses called colonies after about 18 – 24 hours of growth.called colonies after about 18 – 24 hours of growth.

Colonies represent one viable cell or Colony Forming Colonies represent one viable cell or Colony Forming Unit (CFU) that came to rest on the agar surface. Unit (CFU) that came to rest on the agar surface.

This cell or CFU divides many times to form This cell or CFU divides many times to form visible colonies on the agarvisible colonies on the agar

Isolated colonies = Isolated colonies = those not touching other colonies those not touching other colonies

represent clones of the original cell or CFU since represent clones of the original cell or CFU since all the cells in the colony were derived from one cell all the cells in the colony were derived from one cell or CFU and are genetically identical.or CFU and are genetically identical.

Isolated colonies are considered to be pure cultures of Isolated colonies are considered to be pure cultures of a particular bacterial species and strain.a particular bacterial species and strain.

Colony Morphology

Inoculation of a Broth Culture1.1. Label the sterile nutrient broth with the source of the culture, your Label the sterile nutrient broth with the source of the culture, your

initials and the date.initials and the date.2.2. Sterilize a loop in the Bacticinerator.Sterilize a loop in the Bacticinerator.3.3. Using appropriate aseptic technique, remove a loop-ful of broth from Using appropriate aseptic technique, remove a loop-ful of broth from

the mixed culture tube.the mixed culture tube.4.4. Insert the loop into the sterile broth and gently swirl. Retract the loop Insert the loop into the sterile broth and gently swirl. Retract the loop

and sterilize it in the Bacticinerator.and sterilize it in the Bacticinerator.5.5. Incubate the broth at 37 Incubate the broth at 37 C for 24 – 48 hours.C for 24 – 48 hours.6.6. Observe the broth culture for turbidity. Record the results in the Table Observe the broth culture for turbidity. Record the results in the Table

on page 30 in the lab book.on page 30 in the lab book.

Compare your inoculated broth tube to theCompare your inoculated broth tube to theun-inoculated control tube to determine un-inoculated control tube to determine the amount of turbidity. the amount of turbidity.

The more turbid the broth The higher the The more turbid the broth The higher the bacterial count per mL of broth.bacterial count per mL of broth.

Inoculating an Agar Slant

1.1. Label the sterile nutrient agar slant with the source of Label the sterile nutrient agar slant with the source of the culture, your initials, and the date.the culture, your initials, and the date.

2.2. Sterilize the loop using the bacticinerator.Sterilize the loop using the bacticinerator.

3.3. Using appropriate aseptic technique, remove a loopful of Using appropriate aseptic technique, remove a loopful of broth from the mixed culture tube.broth from the mixed culture tube.

4.4. Insert the loop into the sterile agar slant tube and Insert the loop into the sterile agar slant tube and starting at the base of the slant (closest to the bottom of starting at the base of the slant (closest to the bottom of the tube), very lightly draw the loop in a zig-zag motion the tube), very lightly draw the loop in a zig-zag motion up the slant. Do not dig into the agar. Sterilize the loop up the slant. Do not dig into the agar. Sterilize the loop in the bacticinerator.in the bacticinerator.

5.5. Incubate the slant at 37 Incubate the slant at 37 C for 24 – 48 hours.C for 24 – 48 hours.

6.6. In the following lab observe the slant for growth. Record In the following lab observe the slant for growth. Record the results in the table on page 30 in the lab book.the results in the table on page 30 in the lab book.

Streak Plates Allow for the growth of isolated colonies on the surface of the agar.Allow for the growth of isolated colonies on the surface of the agar. Used to isolate clones of a particular bacterial species/strain.Used to isolate clones of a particular bacterial species/strain. An isolated colony, one that is not touching any other colonies, is assumed to An isolated colony, one that is not touching any other colonies, is assumed to

be a pure culture.be a pure culture. May observe colony morphology that can be used to help identify the bacterial May observe colony morphology that can be used to help identify the bacterial

species. species. Colonies of the same organism may grow differently on different media, e.g. Colonies of the same organism may grow differently on different media, e.g.

the shape, color, growth pattern of the colony may differ on other types of the shape, color, growth pattern of the colony may differ on other types of media.media.

Colony Morphology CharacteristicsColony Morphology Characteristics Colony colorColony color Type of hemolysis (if grown on Sheep Blood Agar)Type of hemolysis (if grown on Sheep Blood Agar) FormForm ElevationElevation MarginMargin

Streak Plates

Pour Plating for Colony Counts One of the most common methods of determining cell One of the most common methods of determining cell

number is the viable plate count. number is the viable plate count.

A sample to be counted is diluted in a solution that A sample to be counted is diluted in a solution that will not harm the microbe.will not harm the microbe.

In most cases a volume of liquid from the sample is In most cases a volume of liquid from the sample is first diluted 10-fold. first diluted 10-fold.

In most cases, a 0.1-1.0 ml portion of this first In most cases, a 0.1-1.0 ml portion of this first dilution is then diluted a further 10-fold, giving a dilution is then diluted a further 10-fold, giving a total dilution of 100-fold. total dilution of 100-fold.

This process is repeated until a concentration that is This process is repeated until a concentration that is estimated to be about 1000 cells per ml is reached. estimated to be about 1000 cells per ml is reached.

The sample serially diluted and individual cells are The sample serially diluted and individual cells are deposited in the molten agar and these give rise to deposited in the molten agar and these give rise to colonies.colonies.

By counting each colony, the total number of colony By counting each colony, the total number of colony forming units (CFUs) on the plate is determined. forming units (CFUs) on the plate is determined.

By multiplying this count by the total dilution of the By multiplying this count by the total dilution of the solution (dilution factor), it is possible to find the total solution (dilution factor), it is possible to find the total number of CFUs in the original sample.number of CFUs in the original sample.

Pour Plating for Colony Counts

Calculating CFU from dilution plating results How does a count on a plates get converted to CFUs per gram or ml of How does a count on a plates get converted to CFUs per gram or ml of

sample? Let's illustrate the procedure with an example. Imagine that we sample? Let's illustrate the procedure with an example. Imagine that we perform the following experiment: perform the following experiment:

Five ml of milk are added to 45 ml of sterile broth. From this suspension, two Five ml of milk are added to 45 ml of sterile broth. From this suspension, two serial, 1/100 dilutions are made, and 0.1 ml is plated onto Plate Count Agar serial, 1/100 dilutions are made, and 0.1 ml is plated onto Plate Count Agar from the last dilution. After incubation, 137 colonies are counted on the plate.from the last dilution. After incubation, 137 colonies are counted on the plate.

This problem may be illustrated as follows:This problem may be illustrated as follows:

The initial dilution is calculatedAs follows:

So the initial dilution is 1/10 or 0.1 or 10-1.

Remember, there are many ways to make 1/10 and 1/100 Remember, there are many ways to make 1/10 and 1/100 dilutions. dilutions.

A 0.1 ml to 0.9 ml dilution is the same as a 1 ml to 9 ml A 0.1 ml to 0.9 ml dilution is the same as a 1 ml to 9 ml dilution and a 13 ml to 117 ml dilution. dilution and a 13 ml to 117 ml dilution.

Next, 1 ml of the first dilution is added to 99 ml to make the Next, 1 ml of the first dilution is added to 99 ml to make the second dilution, that is a 1/100 dilution.second dilution, that is a 1/100 dilution.

This is repeated with third dilution giving another 1/100 This is repeated with third dilution giving another 1/100 dilution. dilution.

Then 0.1 ml of the third dilution is plated out on a sterile Then 0.1 ml of the third dilution is plated out on a sterile plate.plate.

The total dilution may be calculated mathematically as follows:

To obtain the concentration of bacteria in the original To obtain the concentration of bacteria in the original sample the “dilution factor” must be determined and then sample the “dilution factor” must be determined and then multiplied by the plate colony count.multiplied by the plate colony count.

The dilution factor is the inverse of the total dilution – The dilution factor is the inverse of the total dilution –

Therefore, the “Dilution factor for our example is: Therefore, the “Dilution factor for our example is: 10106 6 and the and the

Total Colony Forming Units (CFUs) for this Total Colony Forming Units (CFUs) for this sample is:sample is: