MicroBio

Filtration Methodology in Microbiology

  • Filtration Process

    • A sample is filtered through a membrane filter with pores too small for bacteria to pass.

    • The process involves filtering a known liquid sample through a special filter paper.

    • Bacteria and cells are retained on the filter paper, while a sterile liquid passes through.

    • After filtration, the filter paper is placed on an agar medium to allow colonies to grow over 24 to 48 hours.

    • Once colonies grow, enumeration takes place.

  • Example of Filtration

    • Sample Type: Seawater, with a known volume of 100 mL filtered.

    • Outcome: After incubation, 20 colonies were observed.

    • Calculation of CFU (Colony Forming Units):

    • CFUs per 100 mL: [ CFU = \frac{20}{100} = 0.2 \text{ CFUs/mL} ]

    • Proper display: 20 CFUs per 100 mL.

Media Characteristics

  • MacConkey Agar

    • Purpose: Selective for Gram-negative bacteria; differentiates lactose fermenters.

    • Growth Indicators:

    • Lactose fermenters (e.g., E. coli) appear red due to acid production.

    • Non-fermenters (e.g., Salmonella) appear colorless.

    • CFU Calculation:

    • After filtering 100 mL of sample, if 10 red colonies and 5 colorless colonies were observed:

    • For lactose fermenting bacteria: [ CFU = \frac{10}{100} = 0.1 \text{ CFUs/mL} ]

    • Thus, 10 CFUs per 100 mL saline water.

    • Recall: Total reflects that both types observed are Gram-negative as both colonies grew on MacConkey agar.

Most Probable Number (MPN) Method

  • Application: Common for analyzing food and water samples.

  • Setup: 3 sets of 5 tubes (1 set: 10 mL, 2nd set: 1 mL, 3rd set: 0.1 mL of sample added).

    • Each set tests for (+) fermentation of sugar, indicated by gas production (airspace in tubes).

  • Results Interpretation: Count positives in each set. For example:

    • First set (10 mL): 4 positives

    • Second set (1 mL): 3 positives

    • Third set (0.1 mL): 1 positive.

  • MPN Calculation: Use a provided statistical table for the combination of observed positives to find MPN index per 100 mL.

    • Example: If the combination is 4-3-1, corresponding MPN index = 33.

  • Other MPN Scenarios:

    • Initial results colored red; after incubation, positive results turned yellow, indicating growth (fermentation).

    • Example Observations:

    • 500 pattern: 5 positives in the first, 0 in the second, 0 in the third = MPN index of 23.

Indirect Growth Measurement

  • Turbidity Method:

    • Measures cloudiness using a spectrophotometer.

    • Relationship: Higher turbidity reflects more bacterial growth.

    • Process:

    • Clear (uninoculated) samples allow light transmission; turbid (inoculated) samples scatter light.

    • Key Definitions: Absorbance relates directly to cell concentration; higher absorbance indicates greater bacterial density.

Bacterial Genetics Overview

  • DNA Structure:

    • Definitions:

    • DNA (deoxyribonucleic acid) vs. RNA (ribonucleic acid):

      • DNA has deoxyribose sugar; RNA has ribose.

      • DNA includes nitrogenous bases: adenine, thymine, cytosine, guanine.

      • RNA includes adenine, cytosine, guanine, uracil (thymine is absent).

    • DNA is double-stranded, forming a double helix structure.

    • Base Pairing:

    • Adenine pairs with Thymine (A-T); Cytosine pairs with Guanine (C-G).

    • Antiparallel nature of strands: one strand runs 5' to 3', the other 3' to 5'.

    • Counting Nucleotides Example: Given a strand with specific nucleotides, the complementary strand can be derived according to base-pairing rules.

Genome Characteristics

  • Genomic Differences:

    • Prokaryotic vs. Eukaryotic Genomes:

    • Prokaryotes (bacteria and archaea) typically have a single, circular chromosome and are haploid.

    • Eukaryotes have multiple linear chromosomes and are diploid.

    • Chromosomal Packaging:

    • Bacterial DNA is supercoiled to fit within the small cell, and eukaryotic DNA is wrapped with histones.

DNA Replication Process in Bacteria

  • Key Terms:

    • Replication Begins: At the origin of replication; proceeds bi-directionally.

    • Enzymes Involved:

    • Helicase: Unzips DNA strands.

    • Primase: Synthesizes RNA primers.

    • DNA Polymerase:

      • DNA polymerase III synthesizes new DNA strands; DNA polymerase I replaces RNA primers with DNA.

    • Ligase: Joins Okazaki fragments.

  • Semi-Conservative Nature: Each of the new DNA molecules contains one old strand and one new strand.

  • Exponential Growth: DNA replication doubles the number of DNA molecules in each cycle [ P = B \times 2^n ] where $P$ is the final count and $B$ is the initial count after $n$ replications.

  • Example Calculation: If the process starts with 1 DNA molecule, after three cycles, the total becomes 8 DNA molecules.

    • Conversely, if you start with 100 molecules: [ P = 100 \times 2^4 = 1600 \text{ after 4 replications} ]