Invaders 09 10-2012

MICROBIOLOGY PRACTICAL

INVADERS Unit :I

Problem :Invaders

Year :2

Date :Tuesday 09.10.2012

Time :09:00-11:00 a.m

OBJECTIVES: Each student should be familiar with the basic techniques for the

identification of medically important microorganisms (Bacteria, Fungi,

Parasites and Viruses):

Edited by: A. Qareeballa

MATERIALS MAY BE POTENTIALLY INFECTIOUS, YOU

ARE EXPECTED TO ADOPT THE UNIVERSAL

PRECAUTIONS (UP)

STATION-1: 1.Observe and discuss the different types of

culture media ROUTINE LABORATORY MEDIA

1. Basal media, e.g. Nutrient agar.

2. Enriched media, e.g. blood agar.

3. Enrichment media, e.g. Selenite F

4. Differential (Indicator media), e.g. MacConkey agar.

5. Selective media, e.g. Campylobacter medium.

6. Transport media

7. Storage media.

8. Leishmania media

9. Anaerobic media, e.g. (Cooked meat &

Thioglycholate media)


TYPES OF CULTURE MEDIA

Media are of different types on consistency and chemical

composition. A. On Consistency:

1. Solid Media.

a. Advantages of solid media:

• Bacteria may be identified by studying the colony character,

• Mixed bacteria can be separated.

b. Solid media is used for the isolation of bacteria as pure culture.

'Agar' is most commonly used to prepare solid media.

• Agar is polysaccharide extract obtained from seaweed.

• Agar is an ideal solidifying agent as it is :

• Bacteriologically inert, i.e. no influence on bacterial

growth,

• It remains solid at 37°C, and

• It is transparent.

2. Liquid Media. It is used for profuse growth, e.g. blood culture in

liquid media. Mixed organisms cannot be separated. Edited by: A. Qareeballa

B. On Chemical Composition :

1. Routine Laboratory Media

2. Synthetic Media. These are chemically

defined media prepared from pure

chemical substances. It is used in

research work


1. BASAL MEDIA.

Basal media are those that may be used for growth (culture)

of bacteria that do not need enrichment of the media.

Examples: Nutrient broth, nutrient agar and peptone water.

Staphylococcus and Enterobacteriaceae grow in these media.

1. ENRICHED MEDIA. The media are enriched usually by

adding blood, serum or egg. Examples: Enriched media are

blood agar and Lowenstein-Jensen media. Streptococci grow

in blood agar media.

2. SELECTIVE MEDIA. These media favour the growth of a

particular bacterium by inhibiting the growth of undesired

bacteria and allowing growth of desirable bacteria.

Examples: MacConkey agar, Lowenstein-Jensen media,

tellurite media (Tellurite inhibits the growth of most of the

throat organisms except diphtheria bacilli). Antibiotic may

be added to a medium for inhibition. Edited by: A. Qareeballa

4. INDICATOR (DIFFERENTIAL) MEDIA.

An indicator is included in the medium. A particular

organism causes change in the indicator, e.g. blood,

neutral red, tellurite. Examples: Blood agar and

MacConkey agar are indicator media.

5. TRANSPORT MEDIA.

These media are used when specie-men cannot be

cultured soon after collection. Examples: Cary-Blair

medium, Amies medium, Stuart medium.

6. STORAGE MEDIA.

Media used for storing the bacteria for a long period of

time. Examples: Egg saline medium, chalk cooked meat

broth.



BASAL MEDIA

Basal media are those that may be used for growth (culture)

of bacteria that do not need enrichment of the media.

Examples: Nutrient broth, nutrient agar and peptone

water. Staphylococcus and Enterobacteriaceae grow in

these media.

ENRICHED MEDIA

Enriched media: Blood and other special nutrients may be

added to general purpose media to encourage the growth of

fastidious microbes. These specially prepared media are

called as enriched media. e.g. Blood agar, Chocolate agar.



Enrichment media Enrichment media: This is a media which promotes

the growth of a particular organism by providing it

with the essential nutrients and rarely contains certain

inhibitory substance to prevent the growth of normal

competitors. e.g. Selenite F broth- this media favours

the growth of Salmonella also prevents the growth of

normal competitors like E. coli . but E. coli do not

perish in the medium but they do not flourish like

Salmonella



Differential Medium:

Culture medium that allows one to distinguish between

or among different microorganisms based on a difference

in colony appearance (color, shape, or growth pattern) on

the medium.

Dyes in the medium (e.g.: eosin/methylene blue in EMB)

or pH indicators change the color of the medium as

sugars in the medium (e.g.: lactose in EMB &

MacConkey's and Mannitol in MSA) are fermented to

produce acid products



Selective Medium:

Culture medium that allows the growth of

certain types of organisms, while inhibiting

the growth of other organisms

Dyes in the medium (e.g.: methylene blue in

EMB & crystal violet in MacConkey

medium) or high salt concentration in the

medium (e.g.: 7% salt in Mannitol Salt

Agar) inhibit the growth of unwanted

microorganisms


TRANSPORT MEDIA

These media are used when specie-men cannot be

cultured soon after collection. Examples: Cary-Blair

medium, Amies medium, Stuart medium.

STORAGE MEDIA

Media used for storing the bacteria for a long period of

time. Examples: Egg saline medium, chalk cooked meat

broth.


NUTRIENT AGAR A general purpose medium which may be enriched with 10% blood or other biological fluid.

Directions

Suspend 28g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilize by

autoclaving at 121°C for 15 minutes.

Description

Nutrient Agar is a basic culture medium used to subculture organisms for maintenance

purposes or to check the purity of subcultures from isolation plates prior to biochemical or

serological tests.

In semi-solid form, agar slopes or agar butts the medium is used to maintain control

organisms.

Nutrient Agar is suitable for teaching and demonstration purposes. It contains a concentration

of 1.5% of agar to permit the addition of up to 10% of blood or other biological fluid, as

required. The medium, without additions may be used for the cultivation of organisms which

are not exacting in their food requirements.

For a medium which is richer in nutrients, see Blood Agar Base.


Formula gm/litre

'Lab-Lemco' powder 1.0

Yeast extract 2.0

Peptone 5.0

Sodium chloride 5.0

Agar 15.0

pH 7.4 +/- 0.2

Blood Agar

Tryptone 15 g

Phytone or soytone 5 g

NaCl 5 g

Agar 15 g

Distilled water 1 liter

Heat with agitation to dissolve agar. Autoclave 15 min at

121°C. Cool to 50°C. Add 5 ml defibrinated sheep red blood

cells to 100 ml melted agar. Mix and pour 20 ml portions into

sterile petri dishes. Final pH of base, 7.3 ± 0.2.


MacConkey Agar

Proteose peptone or polypeptone 3 g

Peptone or gelysate 17 g

Lactose 10 g

Bile salts No. 3 or bile salts mixture 1.5 g

NaCl 5 g

Neutral red 0.03 g

Crystal violet 0.001 g

Agar 13.5 g

Distilled water 1 liter

Suspend ingredients and heat with agitation to dissolve. Boil 1-2 min.

Autoclave 15 min at 121°C, cool to 45-50°C, and pour 20 ml portions into

sterile petri dishes. Dry at room temperature with lids closed. Final pH, 7.1

± 0.2.


SABOURAUD DEXTROSE AGAR

Sabouraud Dextrose Agar is used for the cultivation of fungi.

Product Summary and Explanation

Sabouraud Dextrose Agar (SDA) is used for cultivating pathogenic & commensal fungi and yeasts.

The high dextrose concentration and acidic pH of the formula permits selectivity of fungi. SDA

enhanced with the addition of cycloheximide, streptomycin, and penicillin to produce an excellent

medium for the primary isolation of dermatophytes.

Sabouraud Dextrose Agar is used clinically to aid in the diagnosis of yeast and fungal infections.

Principles of the Procedure

Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue provide the nitrogen and vitamin

source required for organism growth in Sabouraud Dextrose Agar. The high concentration of Dextrose

is included as an energy source. Agar is the solidifying agent.

Formula / Liter

Enzymatic Digest of Casein......................................................5 g

Enzymatic Digest of Animal Tissue...........................................5 g

Dextrose..................................................................................40 g

Agar.........................................................................................15 g

Final pH: 5.6 ± 0.2 at 25°C

Formula may be adjusted and/or supplemented as required to meet performance specifications.

Directions

1.Suspend 65 g of the medium in one liter of purified water.

2.Heat with frequent agitation and boil for one minute to completely dissolve the medium.

3.Autoclave at 121°C for 15 minutes.


Continue SABOURAUD DEXTROSE AGAR

Expected Cultural Response

Cultural response on Sabouraud Dextrose Agar at 25 - 30°C after 2 – 7 days of

incubation.

Microorganism Response

Aspergillus niger ATCC® 16404 growth

Candida albicans ATCC® 10231 growth

Microsporum canis ATCC® 36299 growth

Penicillium roquefortii ATCC® 10110 growth

Trichophyton mentagrophytes ATCC® 9533 growth

Results Yeasts grow creamy to white colonies. Molds will grow as filamentous colonies of

various colors. Count the number of colonies and consider the dilution factor (if

the test sample was diluted) in determining the yeast and/or mold counts per

gram or milliliter of material.


Continue SABOURAUD DEXTROSE AGAR

Expected Cultural Response

Cultural response on Sabouraud Dextrose Agar at 25 - 30°C after 2 – 7 days of

incubation.

Results Yeasts grow creamy to white colonies. Molds will grow as filamentous colonies of

various colors. Count the number of colonies and consider the dilution factor (if

the test sample was diluted) in determining the yeast and/or mold counts per

gram or milliliter of material.

Microorganism Response

Aspergillus niger ATCC® 16404 growth

Candida albicans ATCC® 10231 Growth

Microsporum canis ATCC® 36299 Growth

Penicillium roquefortii ATCC® 10110 growth

Trichophyton mentagrophytes ATCC® 9533 growth


Anaerobic Transport Media Surgery Pack (ATMSP) Anaerobic Transport Medium Surgery Pack (ATMSP) is a mineral salt base semi-solid

media with reducing agents designed as a holding medium for maintaining viability of

microorganisms, especially anaerobic bacteria through collection, transport and

shipment of clinical specimens from a sterile surgical environment. The Anaerobic

Transport Medium Surgery Pack contains buffered mineral salts in a semi-solid media

with sodium thioglycolate and cysteine added to provide a reduced environment.

Resazurin is added as a redox indicator to reveal exposure to oxygen by turning blue.

This product has been prepared to provide an environment, which maintains viability

of most microorganisms without significant multiplication and allows for dilution of

inhibitors present in clinical material. This medium is designed to meet the stringent

viability requirements of obligate anaerobes. All items are supplied with screw caps

containing rubber septa (Hungate caps), which allows for either direct injection of

aspirated clinical material or introduction of tissue samples. The contents and outer

surface of the tube are sterile. This medium is prepared, dispensed and packaged under

oxygen-free conditions to prevent the formation of oxidized products prior to use.


STATION-1:

2. Observe and discuss the atmospheric

conditions (O2 requirement, incubation

temperature and pH) for microorganisms

growth:

• Aerobic

• Anaerobic

• Microaerophilic

• CO2 requirements.


Ordinary Incubator Most of the medically important organisms require

incubation temperature of: 35oC - 37oC


Anaerobic Incubator for anaerobes

Co2 Incubator


Anaerobic Jar Microaerophilic Jar


Candle Jar for CO2


Anaerobic chamber


STATION-1

3. Examine macroscopically:

• Culture plates showing bacterial colonies

• Antimicrobial susceptibilty testing plates

• Fungi (molds) contaminated bread.

• Cultured plates seeded with Candida

species

• Adult parasite worms, e.g. Ascaris

lumbricoides & Fasciola hepatica



NUTRIENT AGAR

Cultured with some

organisms

Notice: Bacterial

colonies


Hemolysis with Blood Agar

SABOURAUD DEXTROSE AGAR

Cultured with Candida species


Molds growth on Sabouraud agar


STATION-2

1.Explain the preparation of bacteriological

smears and stain the provided prepared smear

with Gram’s staining method.

2.Examine microscopically:

• Gram-positive cocci.

• Gram-negative cocci

• Gram-positive bacilli (rods)

• Gram-negative bacilli (rods)

• Yeast cells.

• Protozoa, e.g. Leishmania species.


Basic Stain Mordant Decolorizer: Counterstain:

Crystal violet

or Gentian

violet

Lugol's

iodine

Acetone

or Acetone-

alcohol

or Ethanol

Neutral red

(1% w/v)

or Safranin

or Diluted

carbol fuchsin

Gram staining technique

Required:


PREPARATION OF THE SMEAR

1. A small sample of a bacterial culture is removed from

a culture. In this example it is being taken from a broth

culture of the pure microbe but it could be removed

from a culture on solid medium or from material

containing bacteria eg faeces or soil.

2.The bacterial suspension is smeared onto a clean glass

slide. If the bacteria have been removed from a culture

on solid media or it is from a soil or faeces sample it

will have to be mixed with a drop of bacteria-free

saline solution.

3.The bacterial smear is then dried slowly at first and

then, when dry, heated for a few seconds to the point

when the glass slide is too hot to handle. This fixes ie

kills the bacteria making the slide safe to handle. Care

must be taken not to overheat which will char the cells Edited by: A. Qareeballa

IMPORTANT NOTES:

• Failure to follow these directions may cause

staining artifacts and disrupt the normal

morphology of bacteria and cells.

• To be visible on a slide, organisms that stain by

the Gram method must be present in

concentrations of a minimum of 104 to 105

organisms/ml of unconcentrated staining fluid.

At lower concentrations, the Gram stain of a

clinical specimen seldom reveals organisms

even if the culture is positive.

• Smears that are not properly fixed tend to be

washed away during staining and washing

resulting in the absence of stained bacteria. Edited by: A. Qareeballa

Results

Gram positive bacteria

Dark purple

Yeast cells Dark purple

Gram negative bacteria Pale to dark red

Nuclei of pus cells Red

Epithelial cells Pale red

Gram staining technique Method

1. Fix the dried smear using gentle heat or fix with methanol for 2 minutes. The purpose of

fixation is to preserve microorganisms and prevent smears being washed from slides during

staining.

2. Cover the fixed smear with crystal violet stain for 30-60 seconds.

3. Rapidly wash off the stain with clean water.

4. Tip off all the water, and cover the smear with Lugol's iodine for 30-60 seconds.

5. Wash off the iodine with clean water.

6. Decolorize rapidly {few seconds} with acetone-alcohol. Wash immediately with clean water.

Caution: Acetone- alcohol is highly flammable; therefore use it well away from an open flame.

7. Cover the smear with neutral red stain for 1 minute.

8. Wash off the stain with clean water.

9. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry.

10. Examine the smear microscopically, first with the 40x objective to check the staining and to

see the distribution of material, and then with the oil immersion objective (X100) to report the

bacteria and cells.


STATION-2

2. Examine microscopically Stained smears

of::

• Gram-positive cocci.

• Gram-negative cocci

• Gram-negative bacilli (rods)

• Gram-positive bacilli (rods)

• Yeast cells.

• Protozoa, e.g. Leishmania species.


Amastigote of Leishmania sp.

Promastigote of Leishmania sp.


VERO Cells, normal Infected with herpes B Virus

http://zeltus.eu/mre/photos/vero-a.jpg

http://zeltus.eu/mre/photos/vero-b.jpg

Phase-contrast microscopic image

of Vero cells. Edited by: A. Qareeballa

Invaders 09 10-2012

Education

Transcript of Invaders 09 10-2012