MIDTERM: BACTE LAB

• PURE CULTURE:

  • A culture which contains a single species of microorganism
  • A population of cells arising from a single cell
• CULTIVATION

  • Increasing the population of microorganisms by providing their nutritional and physical requirements
  • VIVO ENVIRONMENT - Process of growing microorganisms in culture by taking bacteria from the infection site.
  • VITRO ENVIRONMENT - by some means of specimen collection and growing them in the artificial environment of the laboratory.
• CULTURE MEDIUM

  • Any nutrient material growth and cultivation of microorganisms in the laboratory
  • composed variety mixture of nutrients: Carbon, Nitrogen, Sulfur, Phosphorus, Hydrogen, Oxygen and Buffer
  • Inhibitory Agents - facilitates isolation of desired organism while suppressing the growth of another organism
• NUTRIENTS

  • Extraxellular substances which provide the cell with materials for building protoplasm and energy generation
• CULTURE

  • Microbes that grow and multiply in or on a culture medium
  • TYPES:

  1. Pure Culture
  2. Mixed Culture
  3. Stock Culture
• PURE CULTURE

  • Composed of only one spp.
• STOCK CULTURE

  • Composed of more than one spp.
• AGAR

  • Sulfated polymer made up of D-galactose, 3-6-anhydro-L-galactose, and D-glucoronic acid and usually derived from red algae.
  • Melt at 80 to 90 degrees and Solidify at 40 to 50 degrees
• 55 to 60 Degrees

  • cooling temperature for distribution of culture medium into petri plates.
• 20 to 25 mL

  • amount of molten agar transferred to a sterile plate.
• LIQUID MEDIUM

  • commonly called broth in the micro lab
  • enable microorg to be recovered in higher numbers
  • used for detecting the presence of low number microorg that may be missed if plated on solid agar only
  • allows growth of aerobes, anaerobes and facultative anaerobes
• LIQUID MEDIUM EXAMPLE:

  1. Nutrient Broth
  2. Brain Heart Infusion
  3. Trypticase Soy Broth
  4. Thioglycollate
• SEMI-SOLID MEDIUM

  • contains 0.5 to 1% agar
  • has the consistency between a liquid and a solid
  • jelly consistency
  • has a lower concentration of agar than solid media
  • commonly used in determining organism motility
  • observed bacterial motility and detect indole and sulfide production
  • EXAMPLE: Sulfide Indole Motility Medium
• SOLID MEDIUM

  • contains 1.5 - 2% of agar
  • A solidifying agent, such as agar, is added in the preparation of solid culture media
  • does not inhibit bacteria or liquify at room temp
  • develop surface colony growth of microorganisms
  • critical for obtaining isolated colonies
• SOLID MEDIUM EXAMPLES:

  1. Triple Sugar Iron Agar
  2. MacConkey Agar
  3. Blood Agar Plate
  4. Chocolate Agar Plate
• SYNTHETIC MEDIUM

  • All components are chemically defined and exact composition of the chemical is known
  • Comercially prepared culture media
  • used for research purposes
  • preferred for the isolation of cyanobacteria and chemoorganotrophs
  • EXAMPLE: BG-11 medium
• NON-SYNTHETIC MEDIUM

  • not all components are chemically defined
  • precise composition of some or all of the nutritive
  • substances used is not known
  • useful for isolation of medically significant bacteria
  • EXAMPLE:

  1. Nutrient Broth
  2. TSB
  3. MAC

  > agar potato infusion ( plant origin ) beef extract ( animal origin ) yeast extract ( microbial origin )
• TISSUE CULTURE MEDIUM

  • contains living tissues
  • used for obligate intracellular bacteria: Rickettsia and Chlamydia

  EXAMPLES:

  • Hela 229 - human cervical tissue (Chlamydia)
  • McCoy and W138 - fibroblast of mouse cell line
  • Embryonated Egg - propagation of RIckettsia
  • Vero Cell - from African Green Monkey
  • A549 - from Lung Carcinoma
  • Hep-2 Cell - from Laryngeal Carcinoma
• SIMPLE MEDIA

  • routinely used in the lab and without additional supplements
  • support growth of most NON-FASTIDIOUS bacteria to grow at natural rates, without providing advantage to any particular bacteria
  • Usually composed of meat and soybean extracts

  EXAMPLES:

  1. Nutient Agar
  2. Nutrient Broth
  3. TSB
  4. Potato Dextrose Agar
• ENRICHMENT MEDIA

  • used to increase the number of microorg with unusual physiological characteristics
  • enhance the growth of particular organisms and supress the growth of normal flora present in specimen
  • contain specific nutrients and without additional supplements
  • incubated for a certain period and then subculture to isolate the desired organism
  • can also be used as a supplement to agar plates to detect aerobes, anaerobes an microaerophiles (THIOGLYCOLLATE)
  • media with additional supplements necessary for the growth of fastidious organisms
  • solid type media
• ALKALINE PEPTONE WATER

  • promotes growth of Vibrio spp. before inoculation into Thiosulfate-Citrate-Bile-Salts Agar
  • Adjusted to pH 8.5
• SELENITE F

  • Isolation of Salmonella from feces, urine, and water sample
• THIOGLYCOLATE

  • general support enrichment medium that promotes the growth of almost all non-fastidious bacteria
  • 0.075% agar
  • Resazurin - oxidation-reduction indicator
  • Thioglycolic Acid - reducing agent
• TETRATHIONATE

  • selective enrichment broth for the isolation of Salmonella and Proteus
  • Bile Salt and Thiosulfate - suppresses the growth of other coliform bacilli
• GRAM NEGATIVE BROTH

  • isolation of Salmonella and Shigella
  • Enrichment and Selective Medium
  • Sodium Citrate and Sodium Deoxycholate - inhibit gram-positive organisms
  • Mannitol - primary carbon source
• LIM BROTH

  • Group B Streptococci
• ENRICHED MEDIA

  • culture media that contains nutrient supplements only

  EXAMPLE:

  1. Blood Agar Plate
  2. Chocolate Agar Plate
• Blood Agar Plate

  • contains 5% defibrinated blood
  • differentiate hemolytic pattern of bacteria
  • Choices of blood

  1. Sheep
  2. Horse
  3. Rabbit
• Chocolate Agar Plate

  • blood has been chemically treated (80degrees) to lyse the RBC
  • Isolation of fastidious microorganisms
  • Neisseria Gonorrhoeae and Haemophillus spp.
• X FACTOR = Hemin
• V FACTOR = Nicotinamide Adenine Dinucleotide
• DIFFERENTIAL MEDIA

  • allow the visualization of metabolic differences between groups of bacteria
  • distinguishes org. growing together by their differences in cultural characteristics.
  • allow grouping of microbes based on different characteristics demonstrated on the medium
  • with specila reagent like pH indicators or dye

  EXAMPLE:

  1. BAP
  2. Eosin Methylene Blue
  3. MacConkey Agar
• Eosin Methylene Blue

  • lactose and sucrose (differs bacteria base on their fermentation
• MacConkey Agar

  • differentiate lactose-fermenter (pink colonies) from non-fermenter
  • COMPONENTS: Lactose and Bile Salts
• 7.2 tp 7.4 - right pH for Tube Method
• MUCOID COLONY

  • exhibits water-like, glistening, confluent appearance
  • characteristic of org. that form slimes or well-developed capsule
• Smooth Colony

  • uniform texture and homogeneity
  • easily emulsified in NSS
• Rough Colony

  • granulated and rough appearance
  • hard to emulsify in NSS
  • usually produced by mutant strains that lack surface proteins or polysaccharides indicating loss of virulence
• Hemolysis

  • observed in the media immediately surrounding or underneath the colony is a reaction caused by enzymatic or toxin activity of bacteria
  • presumptive identification of strepto and entero
• SWARMING

  • described as the formation of concentric zones of bacterial growth, able to cover the whole surface of solid culture medium