General Concepts in Microbiology 2

General Concepts in Microbiology 2. Anchela Y. Uy-Biag, MD, MBA, DPASMAP.

Content Outline. Microbial Growth Media selection and isolation Methods of detection and identification Microscopy and Staining Antigen detection Antibiotic susceptibility.

Microbial Growth.

Microbial Growth. Increase in number of cells, not cell size One cell becomes colony of millions of cells Control of growth is important for infection control growth of industrial and biotech organisms.

Bacterial Division. Bacteria divide by binary fission usually perpendicular to the length axis and thereby two new cells are produced Alternative means Budding Conidiospores (filamentous bacteria) Fragmentation.

07_13.

Generation Time. Time required for cell to divide/for population to double Average for bacteria is 1-3 hours E. coli generation time = 20 min 20 generations (7 hours), 1 cell becomes 1 million cells!.

07_14a. For a unicellular bacterium, the cell number increase exponentially with base 2 as seen in the table below:.

Standard Growth Curve. 07_15.

Phases of Growth. Lag phase - making new enzymes in response to new medium - the cell division is delayed due to how the inoculum has been treated Log phase - exponential growth - as fast as the soluble nutrients permit - the doubling time can be determined here - most sensitive to drugs and radiation during this period.

Phases of Growth. Stationary phase – nutrients becoming limiting or waste products becoming toxic an essential nutrient has ceased pH-changes due to end products death rate = division rate Death phase – death exceeds division due to some toxic substance excreted from the bacteria.

The Requirements for Growth: Physical Requirements.

Temperature Optima. Optimum growth temperature is usually near the top of the growth range Death above the maximum temp. comes from enzyme inactivation Mesophiles most common group of organisms 40 º F (5 ° C) slows or stops growth of most microbes.

Temperature. Figure 6.1. Thermophiles Hyperthermophiles Mesophiles Psychrotrophs Psychrophiles -10 10 20 30 40 50 60 Temperature (oc) 70 80 90 100 110.

pH. - Most bacteria grow between pH 6.5 and 7.5 - Acid (below pH 4) good preservative for pickles, sauerkraut, cheeses - Acidophiles can live at low pH.

The Requirements for Growth: Physical Requirements.

The Requirements for Growth: Physical Requirements.

Nutritional Factors - Chemical Requirements. Carbon Structural organic molecules, energy source Chemoheterotrophs use organic carbon sources Autotrophs use CO 2 Nitrogen In amino acids and proteins Most bacteria decompose proteins Some bacteria use NH 4 + or NO 3 – A few bacteria use N 2 in nitrogen fixation.

Nutritional Factors - Chemical Requirements. Phosphorus In DNA, RNA, ATP, and membranes PO 4 3 – is a source of phosphorus Sulfur In amino acids, thiamine and biotin Most bacteria decompose proteins Some bacteria use SO 4 2 – or H 2 S.

Oxygen Requirements. Obligate aerobes – require O 2 Facultative anaerobes – can use O 2 but also grow without it Obligate anaerobes – die in the presence of O 2.

The Requirements for Growth: Physical Factors - Chemical Requirements.

Trace elements Inorganic elements required in small amounts Usually as enzyme cofactors Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines.

Sporulation / Endospores. Formation of endospores in Bacillus, Clostridium and G+ genera Can Survive long periods of drought Axial nucleoid Endospore septum grows Spore coat and Exosporium Germination- 3 stages: 1) Activation, 2) germination, 3) outgrowth.

Media selection and isolation.

Most common growth media Nutrient broth is in liquid form Agar is a complex polysaccharide that creates a solid medium Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100°C Solidifies ~40°C.

Defined Media Known quantities of all ingredients Provides trace elements and vitamins required by microbes with a defined carbon and nitrogen source Undefined Media Has complex ingredients which consists of a mixture of many chemical species in unknown proportions.

Suppress unwanted microbes and encourage desired microbes..

Make it easy to distinguish colonies of different microbes..

METHODS OF DETECTION AND IDENTIFICATION.

Labs Used in Diagnosis of Infectious Diseases. Morphologic identification of the agent in stains of specimens or sections of tissues Culture isolation and identification of the agent Detection of antigen from the agent by immunoassay.

Labs Used in Diagnosis of Infectious Diseases. DNA-DNA or DNA-RNA hybridization Detection and amplification of organism nucleic acid in patient’s specimens 6. Demonstration of meaningful antibody or cell-mediated immune responses to an infectious agent.

A properly collected specimen is the single most important step in the diagnosis of an infection.

Quantity of material must be adequate. Sample should be representative of the infectious process.

Methods of Identification. Microscopy and Staining Culture systems Antigen detection Molecular diagnostics.

Microscopy and Staining. STAIN REMARKS Gram stain Most widely used Acid-fast stains ( Ziehl-Neelsen , Kinyoun stain) Identify mycobacteria Immunofluorescent antibody (IF) staining Fluorescein-labeled antibodies Used for Bordetella or Legionella.

Gram Staining. Fix smear by heat Cover with Crystal violet Wash with water, do not blot Cover with Gram’s Iodine Wash with water, do not blot Decolorize for 10-30 seconds Wash with water, do not blot Cover for 10-30 seconds with Safranin Wash with water and let dry.

Genus Shape Bacillus Rods Sporolactobacillus Rods Clostridium Rods or filaments Desulfotomaculum Rods or filaments Sporosarcina Cocci in tetrads or packets Thermoactinomycetes Branched filaments.

Culture Systems. MEDIA INDICATION Blood agar (sheep blood) Standard Bordet- Gengou Bordetella spp Chocolate agar Neisseria & Haemophilus MacConkey or EMB Differential media for Enterobacteriacaeae (LF vs NLF) Lowenstein-Jensen Mycobacterium tuberculosis Salmonella-Shigella Agar Salmonella and Shigella.

Antigen/Antibody Detection. Enzyme immunoassays (EIA) Agglutination tests.

Molecular Diagnostics. Nucleic acid probe hybridization of a nucleic acid sequence matched with complementary RNA or DNA Identification using bacterial 16S RNA Labeled probes specific for 16S RNA are mixed Polymerase chain reaction (PCR) Amplify small amounts of DNA using DNA polymerase Identified by labeled probes.

Disk Diffusion Susceptibility Test. Identifies to which antibiotic the organism is susceptible or resistant to.

Minimum Inhibitory Concentration. - Measures the exact concentration of antibiotic necessary to inhibit bacterial growth.

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