Basic Microbiology Practical No. 4

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Questions and Answers

What is the purpose of using a mordant in the Gram staining procedure?

  • To wash away excess stain
  • To enhance the binding of the primary stain (correct)
  • To provide a contrasting color
  • To stain the bacterial cells

Which of the following steps follows the application of crystal violet in the Gram staining process?

  • Pour off the crystal violet stain and wash (correct)
  • Heat-fix the smear
  • Flood the slide with decolorizer
  • Flood the slide with counter stain

At what temperature should the Petri dish be incubated for bacterial culture?

  • 30 °C
  • 25 °C
  • 37 °C (correct)
  • 42 °C

What is the first step in preparing a slide for Gram staining?

<p>Place a small drop of water on the slide (C)</p> Signup and view all the answers

What is an important consideration when heat-fixing a smear?

<p>Overheating can rupture bacterial cells (B)</p> Signup and view all the answers

Which reagent in the Gram staining process acts as a decolorizer?

<p>Acetone or Ethanol (D)</p> Signup and view all the answers

When observing a stained smear under a microscope, which objective lens should oil be used with?

<p>x100 lens (A)</p> Signup and view all the answers

What is the benefit of using an inverted position for incubating the Petri dish?

<p>To reduce evaporation of moisture (C)</p> Signup and view all the answers

In identify bacterial cultures, which of the following describes Staphylococcus aureus?

<p>Gram-positive, cocci, cluster arrangement (B)</p> Signup and view all the answers

What is the purpose of counterstaining in the Gram stain method?

<p>To distinguish between Gram-positive and Gram-negative bacteria (C)</p> Signup and view all the answers

What is the primary purpose of the streak plating technique?

<p>To isolate individual bacterial colonies on an agar surface (B)</p> Signup and view all the answers

Which type of bacteria retains the crystal violet stain during the Gram staining procedure?

<p>Gram-positive bacteria (D)</p> Signup and view all the answers

What are Colony Forming Units (CFU) used to quantify?

<p>The number of viable bacterial cells in a sample (B)</p> Signup and view all the answers

What is the first step in the Gram staining procedure?

<p>Application of crystal violet (C)</p> Signup and view all the answers

What is the primary purpose of the streaking method?

<p>To determine if a culture consists of one or multiple species (B)</p> Signup and view all the answers

What does the term 'pure culture' imply?

<p>A culture that contains one type of bacterial species (A)</p> Signup and view all the answers

Which of the following media is NOT typically used for isolating bacterial organisms?

<p>Gelatin medium (C)</p> Signup and view all the answers

Which of the following techniques involves using a glass spreader?

<p>Spread plate technique (C)</p> Signup and view all the answers

During microscopy, which characteristic helps differentiate between Gram-positive and Gram-negative bacteria?

<p>Color after Gram staining (C)</p> Signup and view all the answers

What should be avoided when performing an isolation streak plate?

<p>Cutting the surface of the agar with the loop (C)</p> Signup and view all the answers

Which staining technique allows for the visualization of specific cellular structures?

<p>Selective stains (B)</p> Signup and view all the answers

What precaution should be taken while processing samples in a microbiology lab?

<p>Wear appropriate personal protective equipment (B)</p> Signup and view all the answers

What technique is used to quantify bacterial colonies after the spread plating method?

<p>Colony count assessment (C)</p> Signup and view all the answers

What is the result of a perfect spread plate technique?

<p>Visible and countable evenly distributed colonies (C)</p> Signup and view all the answers

What structural feature differentiates Gram-negative bacteria from Gram-positive bacteria?

<p>Presence of an outer membrane (C)</p> Signup and view all the answers

Which type of staining uses dyes that stain only the background?

<p>Indirect or negative staining (A)</p> Signup and view all the answers

What is a key characteristic of Gram-positive bacteria?

<p>They have a thick peptidoglycan layer (C)</p> Signup and view all the answers

What is the best position for incubating agar plates?

<p>Upside down position with lids on (C)</p> Signup and view all the answers

Which of the following is true about indirect staining?

<p>It highlights certain structures like capsules (B)</p> Signup and view all the answers

What role does microscopy play in microbiology?

<p>It helps in identifying and observing microbial morphology and behavior (A)</p> Signup and view all the answers

What is the primary purpose of Gram staining?

<p>To differentiate between Gram-positive and Gram-negative organisms (B)</p> Signup and view all the answers

Which type of bacterial cell wall retains the crystal violet-iodine complex during Gram staining?

<p>Gram-positive cell wall (B)</p> Signup and view all the answers

What color do Gram-negative bacteria appear after the Gram staining procedure?

<p>Pink to red (C)</p> Signup and view all the answers

In the streak plating technique, what is the purpose of sterilizing the inoculation loop after streaking?

<p>To avoid cross-contamination during subsequent streaks (B)</p> Signup and view all the answers

How should the Petri dish be manipulated during the streak plating technique?

<p>Turn it so the opposite end becomes the starting point for new streaks (C)</p> Signup and view all the answers

What type of agar plates were used in the isolation of pure cultures in the practical case?

<p>Blood agar and MacConkey agar plates (C)</p> Signup and view all the answers

Which of the following best describes the staining property of Gram-positive organisms?

<p>They retain the crystal violet and do not take up the counterstain (B)</p> Signup and view all the answers

What is the composition of the crystal violet-iodine complex during Gram staining?

<p>A combination of crystal violet dye and an iodine solution (C)</p> Signup and view all the answers

At what temperature and conditions is incubation typically done for the culture of bacteria in this method?

<p>37°C in aerobic conditions (C)</p> Signup and view all the answers

Which microscopy technique can be used to observe stained bacterial cultures?

<p>Bright field microscopy (C)</p> Signup and view all the answers

Flashcards

Pure Culture

A culture containing only one type of bacteria.

Isolation Technique

Methods used to separate one type of bacteria from a mixed culture.

Streak Plate

A technique to isolate bacteria by spreading a sample across an agar plate.

Streaking

The act of spreading a sample across an agar surface using an inoculation loop.

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Agar Plate

A solid surface used for growing bacteria.

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Colony-Forming Unit (CFU)

A single bacterial cell or group of cells that form a visible colony.

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Mixed Culture

A culture containing more than one type of bacteria.

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Inoculation loop

A tool used for transferring bacteria.

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Microorganism

An organism too small to be seen with the naked eye.

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Culture

Microorganism grown in a lab to study it.

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Gram Staining

A lab technique to categorize bacteria (Gram-positive and Gram-negative) based on cell wall structure

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Gram-positive bacteria

Bacteria with thick cell walls that retain crystal violet dye during staining.

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Gram-negative bacteria

Bacteria with thin cell walls that do not retain the crystal violet dye.

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Peptidoglycan

A compound in bacterial cell walls that determines staining properties.

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Crystal Violet

A primary stain in Gram staining that initially colors both Gram-positive and Gram-negative.

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Counterstain

A second dye, like safranin, that stains Gram-negative cells after decolorization.

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Pure Culture Isolation

The process of growing only one type of bacteria from a sample.

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Blood Agar Plate

A lab plate used to grow bacteria with a combination of nutrient and blood.

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MacConkey Agar

A lab plate selective for certain types of bacteria based on their ability to ferment lactose.

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Streaking Method

A technique used to isolate individual bacterial colonies on an agar plate.

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Streak plate method

A microbiological technique used to isolate individual bacterial colonies from a mixed culture.

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Mixed/Contaminated Culture

A culture containing more than one type of microorganism.

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Viable Culture

A culture that is able to grow.

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Direct Staining

Staining technique where aniline dyes directly stain bacterial cells.

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Indirect/Negative Staining

Staining technique that stains the background, leaving the microorganisms unstained. Used to see capsules or spores.

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Selective Staining

Staining technique for specific parts of an organism (spor, granules, flagella).

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Differential Staining

Staining technique used to distinguish different groups of bacteria (e.g., Gram-positive vs. Gram-negative).

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Spread Plate Technique

Technique of spreading a liquid sample or culture evenly on an agar plate.

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Microbial Staining

Technique used to view microbial cells under a microscope, as they are too small to see with the naked eye.

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Primary Stain

The first dye applied in Gram staining, which stains all bacterial cells the same color, usually violet.

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Mordant

A reagent used in Gram staining to intensify the primary stain and make it more difficult to remove from Gram-positive bacteria.

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Decolorizer

A reagent used in Gram staining to remove the primary stain from Gram-negative bacteria.

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Heat-Fixing

A step in slide preparation where the smear is briefly passed over a flame to kill the bacteria and attach them to the slide.

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Smear Preparation

The process of spreading a bacterial sample thinly on a microscope slide for visualization.

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Microscope Observation

The process of examining the stained smear under a microscope to observe the bacterial morphology and Gram reaction.

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Study Notes

Basic Microbiology - Practical No. 4: Isolation of Pure Culture and Gram Staining

  • Intended Learning Objectives (ILOs):
    • Define pure culture and methods for obtaining it
    • Explain the purpose of different isolation techniques
    • Demonstrate the isolation streak plate procedure
    • Prepare bacterial smears and perform Gram stain
    • Explain the staining technique's chemical basis and principle
    • Differentiate Gram-positive and Gram-negative bacteria using a light microscope
    • Describe the differences in cell wall structures of Gram-positive and Gram-negative cells
    • Understand the use of different media for isolating various bacterial organisms

Precautions

  • Wear appropriate personal protective equipment (PPE)
  • Adhere to Standard Operating Procedures (SOPs) for sample processing
  • Immediately record results

Introduction/Background

  • Culture: Cultivating microorganisms in a lab for study
  • Crucial to ensure samples are pure cultures (single type of bacteria) or not mixed
  • Mixed cultures often come from non-sterile sources

Methods of Isolation of Pure Culture

  • Streak plating: Using a sterile inoculation loop to spread a sample across an agar plate
  • Spread plating: Using a glass spreader to spread a liquid sample evenly on an agar plate
  • Pour plating: Mixing diluted samples with melted agar, then pouring into sterile plates

Streak Plate Technique

  • Technique isolates individual bacterial colonies (CFU) on an agar plate using a wire loop
  • Aims to: differentiate pure and mixed cultures; identify viable cultures

Spread Plate Technique

  • Technique where a sample is placed on a plate, then spread evenly using a spreader
  • Ensure even and visible colonies

Microbiological Staining

  • Microbial cells are too small to see without a microscope
  • Staining enhances visibility by giving them colour
  • Direct staining: using aniline dyes to stain bacterial cells directly. Exceptions include bacterial spores and those with a waxy coating
  • Indirect/Negative staining: Staining the background to visualize bacteria/structures

Gram Staining

  • Developed by Hans Christian Gram (1853–1938)
  • Differentiates bacteria as Gram-positive or Gram-negative based on cell wall composition
  • Purpose: Differentiate Gram-positive and Gram-negative organisms, and learn about their cellular morphology & arrangements
  • Principle: Gram-positive cells retain crystal violet-iodine complex; Gram-negative cells decolorize, allowing a counter stain (safranin) to bind
  • Gram-positive cells appear blue/purple; Gram-negative appear pink/red

Preparation & Staining Steps (Detailed in separate pages 7-8)

  • Preparing bacterial smears
  • Applying specific stains
  • Avoiding excessive heat during heat fixation
  • Using various solutions: Crystal violet, Gram's iodine, alcohol decolorizer and counter stain like safranin

Observation

  • Record Gram reactions, shapes and arrangements for observed samples (e.g., samples A and B)

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