PHT 416 Lab# 4 Culture mediaCulture media Streaking Streaking.

PHT 416 Lab# 4PHT 416 Lab# 4

•Culture mediaCulture media• StreakingStreaking

Culture MediaCulture Media

The culture mediaThe culture media is an artificial is an artificial preparation contains the preparation contains the essential element and nutrient in essential element and nutrient in a proper concentration needed a proper concentration needed by the microorganism to grow.by the microorganism to grow.

It may be:-It may be:-Liquid (broth)Liquid (broth)Solid (containing agar)Solid (containing agar)Semisolid (containing low conc of agar)Semisolid (containing low conc of agar)

Culture MediaCulture Media

Most common ingredients:-

1. Essential elements and nutrients.

2. Solidifying agents.

Objectives of bacterial culture:Objectives of bacterial culture:

To studyTo study bacterial morphology, bacterial morphology, physiology and pathogenic physiology and pathogenic propertiesproperties..

At the clinical level:At the clinical level:1.1.Isolation of bacteria in pure cultureIsolation of bacteria in pure culture

2.2.IdentificationIdentification3.3.Antimicrobial susceptibilityAntimicrobial susceptibility

Preparation of important bacterial Preparation of important bacterial products, e.g., toxins, antigens, products, e.g., toxins, antigens, vaccines … etc.vaccines … etc.

Essential Elements and NutrientsEssential Elements and Nutrients

1.1. A Source of Nitrogen:A Source of Nitrogen: • (peptone) (peptone) • meat extract,meat extract,• yeast extract, yeast extract, • amino acids & in organic amino acids & in organic

compound.compound.


2.2. A Source of Carbon:A Source of Carbon: • usually supplied in form of usually supplied in form of

carbohydrates.carbohydrates.

3.3. Inorganic salts:Inorganic salts: • normally present in peptone normally present in peptone

but added in traces [e.g; Na, K, but added in traces [e.g; Na, K, Ca, Mn, Fe, Co, Zn…….etc] Ca, Mn, Fe, Co, Zn…….etc]

♙ NaClNaCl


4. Buffers: added to resist any changed in

pH of the medium.

Blood and milk are naturally occurring buffers.

Solidifying AgentsSolidifying Agents

1. Agar: it is the most common solidifying agent, used in conc. * 1.5 – 2% ( solid medium). * 0.2 – 0.5% (semisolid).

✍ it extracted from certain red marine algae, and composed of complex carbohydrates not easily broken down by most common bacteria.

Solidifying AgentsSolidifying Agents

2.2. Gelatin:Gelatin: it is protein derivative added to it is protein derivative added to

nutrient broth in conc. of 12 -15% nutrient broth in conc. of 12 -15% as a test for proteolytic activity of as a test for proteolytic activity of bacteria. bacteria.

Classification of MediaClassification of Media

According to its content:-According to its content:-

1.1. Non synthetic complex medium;Non synthetic complex medium; • contains extracts or digests of contains extracts or digests of

plant or animal tissues such as plant or animal tissues such as beef, peptone & soybean beef, peptone & soybean digest.digest.


2.2. Synthetic Chemically defined Synthetic Chemically defined medium;medium;

• contains known chemical contains known chemical compounds such ascompounds such as::

amino acids, amino acids, sugarsugar,, Vitamins & salts.Vitamins & salts.


3.3. Living medium;Living medium; • it is a living cell of animal or it is a living cell of animal or

plant in which m.o could be plant in which m.o could be cultivated such as viruses cultivated such as viruses (intracellular tissue culture.)(intracellular tissue culture.)

Types of culture media:Types of culture media:

Simple mediaSimple mediaEnriched mediaEnriched mediaSelective mediaSelective mediaIndicator mediaIndicator mediaSelective and indicator mediaSelective and indicator media

Classification of MediaClassification of Media According to the purpose of

use:-1- Simple [Basic] media; • contain only the essential or

basic nutrients which support the growth of most common bacteria.

✎ e.g; • Nutrient agar (plate, slant)• Nutrient broth


2- Enriched media;2- Enriched media; in addition to basic nutrients, it in addition to basic nutrients, it

contains enriched substances contains enriched substances such as blood, serum, vitamins such as blood, serum, vitamins to support the growth of most to support the growth of most pathogens can’t grow on pathogens can’t grow on ordinary simple media.ordinary simple media.

✎✎ e.g; e.g; blood agar, blood agar, Chocolate agar, Chocolate agar, Serum agar.Serum agar.

Blood agarBlood agar

Chocolate agarChocolate agar(Neisseria spp.)(Neisseria spp.)

C.diphtheria


3. Selective media; mostly used in case of mixture. Contains substances which selectively

enhance the growth of certain particular species and inhibit the growth of undesired bacterial species.

These inhibitory substances include: dyes, heavy metals, chemicals, antimicrobial agents …

Examples of Selective MediaExamples of Selective Media

1.1. Mannitol salt agar;Mannitol salt agar; • it is selective medium for it is selective medium for

Staphylococcus speciesStaphylococcus species because it contains high conc. because it contains high conc. of salt (about 7%).of salt (about 7%).

2- 2- Lowenstein Jensen medium it is selective medium for it is selective medium for M.

tuberculosis;; • because it contains malachite because it contains malachite

green dye.green dye.• It contains also eggs which It contains also eggs which

solidify the medium without solidify the medium without agar and make it enriched agar and make it enriched medium. medium.


3- MacConkey agar:3- MacConkey agar: • it is selective medium for it is selective medium for gram gram

–ve–ve bacteriabacteria because it contains because it contains bile saltbile salt which inhibit the which inhibit the growth of gram +ve bacteria.growth of gram +ve bacteria.


4- Cetrimide agar: it is highly selective medium for

pseudomonas species. It contains Cetrimide.

Cetrimide is a surfactant that inhibit the growth of all organism except pseudomonas.


4- Indicator media:4- Indicator media: Contain substances which are Contain substances which are

affected by the growth of affected by the growth of organism to produce organism to produce characteristic reaction such as characteristic reaction such as indicators which indicates acid indicators which indicates acid production during sugar production during sugar fermentationfermentation..

Examples of Diagnostic MediaExamples of Diagnostic Media

1.1. Mannitol salt agar;Mannitol salt agar; It contains mannitol and phenol red It contains mannitol and phenol red

indicator. indicator. S.aureusS.aureus

is the only strain of staphylococcus is the only strain of staphylococcus species can ferment mannitol, species can ferment mannitol, leading to production of acid which leading to production of acid which can detected by yellow color can detected by yellow color around the growth resulting from around the growth resulting from changing the color of the indicator. changing the color of the indicator.

Growth with yellow color around the colonies S.aureus

Growth without change in the color of the medium S.epidermidis

Examples of Diagnostic MediaExamples of Diagnostic Media

2.2. MacConkey agar;MacConkey agar;

Gram –ve bacteria classified to:Gram –ve bacteria classified to:

It contains neutral red indicator which It contains neutral red indicator which detect acid production due to fermentation detect acid production due to fermentation of lactose which done by lactose fermenter of lactose which done by lactose fermenter gram –ve bacteria such as E-coli and gram –ve bacteria such as E-coli and Klebsiella species to give rose pink colonies. Klebsiella species to give rose pink colonies.

Lactose fermenter Lactose non-fermenter

Lactose fermenter Pink colonies

Lactose non-fermenter Pale colonies


5.5. Selective and indicator media;Selective and indicator media; are are both selective and both selective and

diagnosticdiagnostic since they permit since they permit the growth of a few desired the growth of a few desired species only & contain indicator species only & contain indicator which enable us to differentiate which enable us to differentiate between the species which are between the species which are able to grow on these media.able to grow on these media.

e.g.; Mannitol salt agar, e.g.; Mannitol salt agar, MacConkey agar.MacConkey agar.

Media for FungiMedia for Fungi

❀❀ Sabouraud dextrose agar.Sabouraud dextrose agar.

similar in composition as those of similar in composition as those of bacteria bacteria EXCEPT:EXCEPT:

1.1. They are acidic in nature (PH 3 -6).They are acidic in nature (PH 3 -6).

2.2. contain readily fermentable contain readily fermentable sugars e.g.; dextrose and sugars e.g.; dextrose and maltose.maltose.

☞ ☞ Culture of Fungi usually incubate Culture of Fungi usually incubate at lower temperature than at lower temperature than bacteria (about 25°C). bacteria (about 25°C).

It is anaerobic medium due to presence of:It is anaerobic medium due to presence of: Sodium thioglycolate and cystaine which act Sodium thioglycolate and cystaine which act

as reducing agents.as reducing agents.

Fluid thioglycolate:Fluid thioglycolate:

Anaerobic mediaAnaerobic media

It is anaerobic medium due to presence of:It is anaerobic medium due to presence of: Meat particles (prepared from heart Meat particles (prepared from heart

muscles) muscles) Hematin& glutathione in meat particles Hematin& glutathione in meat particles

act as reducing agents.act as reducing agents.

Cooked meat medium:Cooked meat medium:

Anaerobic mediaAnaerobic media

Anaerobic Jar - The nutrient agar plates employed for routine culturing do not contain reducing agents. - They can be used to culture anaerobes if they are placed inside an anaerobe jar ( a chamber from which the oxygen is removed). - Gas generator packets (e.g. GasPak) are placed in the jar along with plates or tubes containing conventional media destined to be incubated anaerobically. - In order to assure that oxygen actually was removed from the chamber, a strip of paper soaked in methylene blue dye is included in the jar. - It is blue when exposed to oxygen but will become colorless (white) when oxygen is absent.

GAS PAK SYSTEM FOR ANAEROBIC CULTIVATION

Colony vs. CellColony vs. Cell

Colonies Cells

Introduction to Microbiological Introduction to Microbiological Equipments and MaterialsEquipments and Materials

Inoculation:Inoculation: Culturing of sterile Culturing of sterile media with m.o [Inoculation loop].media with m.o [Inoculation loop].

Incubation:Incubation: Placing the culture into Placing the culture into the incubator at optimum temperature the incubator at optimum temperature for growth.for growth.

Sterile:Sterile: Free from any living Free from any living microorganismmicroorganism

Contamination:Contamination: Introduction of Introduction of undesirable m.o.undesirable m.o.

Description of ColoniesDescription of Colonies

Isolation of Pure Colonies Isolation of Pure Colonies of Microorganism of Microorganism

“Streak Plate Method”“Streak Plate Method”In natural environments, bacteria & In natural environments, bacteria &

other m.o exist in mixed other m.o exist in mixed population.population.

““Streak Plate Technique”Streak Plate Technique”The individual cells are separated from each other by certain distance on the surface of the agar.After incubation, each single deposited cell divide many times and finally form visible mass of growth “COLONY”.

The streak Plate MethodThe streak Plate Method

The culture prepared from a The culture prepared from a single type of colony is regarded single type of colony is regarded as a as a pure culturepure culture..

The streak Plate Method is used The streak Plate Method is used for:for: Checking the purity of a bacterial Checking the purity of a bacterial

culture.culture. Isolating individual species from a Isolating individual species from a

mixture culture.mixture culture.


Objective:-Objective:-

for isolation of individual for isolation of individual species of a mixed broth culture.species of a mixed broth culture.

Materials:-Materials:- Nutrient agar plate.Nutrient agar plate. Mixed broth culture of Mixed broth culture of Serratia Serratia

marcescens marcescens andand staph. aureus. staph. aureus.


ProcedureProcedure::

S & SS & S

Aseptic technique Aseptic technique

Invert the plate and Invert the plate and Incubate for 24h at 37Incubate for 24h at 37℃℃

Drop of the cultureDrop of the cultureFlame & Cool

Flame & Cool

Flame & Cool

Sources of Sources of ContaminationContamination

Objective:Objective: To identify some of the To identify some of the sources of contamination present in the sources of contamination present in the lab. lab.

✔✔ in order to avoid them in order to avoid them Contamination from hands.Contamination from hands. Contamination from breath.Contamination from breath. Contamination from air.Contamination from air. Contamination from bench.Contamination from bench.


Sterile nutrient agar plateSterile nutrient agar plate a-a- unwashed unwashed

b-b- washed with water washed with water

c-c- disinfected with alcohol disinfected with alcohol

d-d- control control incubate the plate Inverted at 37°c incubate the plate Inverted at 37°c

for 24 hr.for 24 hr. Record the appearance of the plateRecord the appearance of the plate

a b

c d

1.Contamination from hands:


2. Contamination from breath:2. Contamination from breath: Take a sterile nutrient agar Take a sterile nutrient agar

plate plate Hold it in front of your mouthHold it in front of your mouth Cough and breath vigorouslyCough and breath vigorously Invert the plate and incubate Invert the plate and incubate

for 1 day at 37 °cfor 1 day at 37 °c Record the appearance of the Record the appearance of the

plate.plate.


3.Contamination from air:3.Contamination from air: Expose one sterile Expose one sterile

nutrient agar plate on nutrient agar plate on the bench for 30 minthe bench for 30 min

Invert the plate and Invert the plate and incubate for 1 day at 37 incubate for 1 day at 37 °c°c

Record the colonial Record the colonial appearance appearance 30 min


4.Contamination from bench:4.Contamination from bench: Take a sterile nutrient agar Take a sterile nutrient agar

plate and mark out 2 sections plate and mark out 2 sections on its baseon its base

Take a swab from unclean part Take a swab from unclean part of the bench and press it over of the bench and press it over one sectionone section

Take another swab from Take another swab from cleaned part with disinfectant cleaned part with disinfectant and press it over the second and press it over the second sectionsection

Invert the plate and incubate Invert the plate and incubate for 1 day at 37 °cfor 1 day at 37 °c

Record the result.Record the result.

uc

c

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Thank you

PHT 416 Lab# 4 Culture mediaCulture media Streaking Streaking.

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