Biological Safety Cabinet

- Are hood, that are type of containment barrier that protects the
worker from aerosolized transmission of organism.

Classes

BSC Class I

- Uses exhaust fan to move air ( Unsterilized ) inward the open front

- Air is circulated within the BSC

- Passes through HEPA ( High Efficiency Particulate Air ) filter before

reaching the outside environment

o HEPA filter – filters microorganisms with greater 0.3 um

BSC Class II

- Most common in laboratory laboratories

- Air is pulled inward & downward by a blower

- Passes through HEPA filter for sterilization before reaching the work
surface

- A % of air is HEPA filtered before exhausting outside the environment

o Aka Vertical Laminar Air Flow – air inside form sheaths

BCS Class III

- Self-contained ventilated system

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- Uses for highly infectious microorganism

- Provide the highest level of protection

- Closed front w/c contained already gloves for manipulation of work

surface

Subclasses

Biosafety levels

- Levels of containment. Each level of containment describes the

microbiological practices, safety equipment, facility safeguards for the
corresponding level of risk associated with particular agent

Biosafety Level I

- Agents that are not classified & not known to caused disease
consistently in health adults

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- Laboratory work can be conducted on open bench tops in BSL-1 lab

- Standard microbiology practices should be followed at all times

- Examples of BSL I organism: Bacillus subtillis, Enterobacter aerogenes,

Naegleria gruberi

Biosafety Level II

- Agents that pose moderate potential hazard for employees and the
environment

- Agents most commonly bring sought in clin specimen & used in

diagnostic teaching in other lab

- Includes all agents of infectious dse

- Examples of BSL 2 organism: HIV, HBV, Salmonella spp., Toxoplasma

grundii, prions, abnormal proteins (spongiform encephalitis)

Biosafety Level III

- Infectious agents that either indigenous or exotic

- Potential for aerosol transmission & disease may have serious lethal
consequences

- Organism that are unlikely to be encountered in routine clin lab

- Examples of BSL 3 organism: Mycobacterium tuberculosis, Doxella

burnetti, mold stage of systemic fungi

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o D. burnetti – rickettsia organism that transferred in vectors or
airborne
Biosafety Level IV

- Agents that are dangerous & exotic, high risk of causing life-
threatening infections (no vaccine/ therapy available)

- Can be transmitted by aerosols or have unknown risk of transmission

- BSL 4 facility is either located in separate building or in isolated zone

within the building

- Examples of BSL 3 organism: Marburg virus, Congo-Crimean hemorrhagic

fever virus

Culture Media

Culture

- Microbes that grow & multiply in or on a culture medium

Types of Culture

Mixed Culture

- Contains two species or genera

Pure/Axenic Culture

- Grow one species or genera only

Stock culture

- For future uses/ further studies

Culture medium

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- A medium for growing microorganism that contains essential nutrients
in proper concentration adequate amount of salt water supply free of
inhibitory substances desired consistency, proper pH & is sterile
- A liquid, semi-solid, or solid preparation utilized to observe growth of
microorganism as well as transport and storage

Food Elements

- Peptone – protein
- Carbohydrates – energy
- Minerals – vitamins (low conc allow bacterial growth only: K, Mg, Fe)
- Meat/ Beef extract
- Yeast Extract – sources of amino acids, Vit B (Saccharomyces
cerevisiae)
- Bile & Bile salts – deoxycholic acids (inhibit growth of gram +)
- Agar & Agarose – main component of culture media from red algae

o Agarose – purified from agar ( gelidium and gracilaria)

Characteristics of agar/agarose

 Meting point – 100 deg

 Solidifying temp – 45 deg
 pH – 7 to 7.5

Classification of Culture media

According to physical state/consistency

- Solid – 2 to 3 % of agar (BAP, MAC, TSI)

- Semi-solid – 0.5 to 1 % (Sulfide Indole motility, decarboxylase media)
- Liquid – broth w/o agar

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According to physical state/consistency

- Synthetic/ Chemically-defined
 Exact composition is known

- Non-Synthetic/complex
 At least 1 component is not chemically defined

- Living state/Tissue
 Composed of living cells for Chlamydia Rickettsia and
virus (M. lepreae cause Hansen’s dse culture in footpads)

According to manner of distribution

- Tube
o Broth
o Butt
o Slant (Simon’s Citrate)

- Plated
o Single
o Double

According to use

Simple

- Routinely used in the laboratory without added supplement (plain)

- E.g. Nutrient agar, Nutrient Broth, Trypticase soy agar, Trypticase soy
broth

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Maintenance

- Support the growth of the microorganism while in storage

- E.g. Brain heart Infusion broth, Nutrient agar slant

Transport

- Support the growth of the microorganism while in transit

- E.g. Carry Blair Medium (stool) , Stuart’s medium

Enrichment

- Used to propagate the growth of certain groups of organisms contain

specific nutrients

- E.g. Alkaline Peptone water – Vibrio spp

Selenite F broth & tetrathionate broth - salmonella

Differential

- Allow visualization of the metabolic differences between bacterial

groups
- E.g. MAC – differentiate lactose
Blood agar plate -differentiate through hemolytic pattern

o Beta Hemolysis - Complete Hemolysis

o Alpha Hemolysis – Incomplete Hemolysis (greenish)
o Gamma Hemolysis – no hemolysis

Selective

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- Incorporated w/ antibiotics or inhibitory substrate to inhibit the
growth of other organisms while promoting the growth of the desired
organism

- E.g. MAC, Bismuth sulfide agar, Xylose-Lysine deoxycholate, Mannitol

Salt agar (7.5% salt – staph)

Inhibitory substances

Inhibits Gram +
- Crystal violet, Basic fuchsin, Bile salts

Inhibits Gram –
- Potassium tellurite, sodium azide

Inhibits swarming of proteus

- Alcohol, choral hydrate

Enrich

- Added supplement necessary for growth of certain group organisms

(fastidious): basal medium added w/ blood serum, vitamins, amino
acids
- E.g. BAP, Chocolate agar (lysed blood)

Special

- Isolation of specific type of organism (LJ medium) TCBS agar

- Lowenstein-Jensen medium for M. tuberculosis

- Middlebrook 7H10 M. tuberculosis

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- Thiosulfate Citrate Bile Salts Sucrose Agar for vibrio

Anaerobic media

- Used to grow anaerobic organisms

- Robertson’s cooked meat media, Thioglycolate broth (indicator:
Resazurin <color pink>)

Medium for Susceptibility Testing

Mueller Hinton Agar

- Agar depth: 4 mm
- pH: 7.2 to 7.4

 Kirby-Bauer = disk diffusion (zone of inhibition)

 E-test

Medium for Biochemical Testing

- Identification of organism based on biochemical reaction

- TSI, Urea broth

o Slant – ability to ferment lactose/sucrose

 Yellow – ferments
 Red – not ferment
o Butt – ferment glucose