Laboratory Assignment: The Following Questions Are From Two

Laboratory Assignmentthe Following Questions Are From Two Exercises D

Laboratory assignment The following questions are from two exercises (dilutions/pipetting and counting bacteria) that you read/studied. As reference materials, read lab manual, lab exercise PPts and/or study guides and answer questions given below fully/completely in the spaces provided as instructed. Please submit assignment before or on the due date. Handwritten answers are not acceptable, except calculations. You may submit assignment as an attachment to an email. Alternatively, you may scan assignment or take a photo of it and submit as an attachment to an email.

Paper For Above instruction

The laboratory assignment encompasses two key exercises: dilutions/pipetting and bacteria counting, which are fundamental skills in microbiology laboratory practice. These exercises aim to enhance the student's understanding of core procedures used to prepare serial dilutions and accurately measure bacterial populations in various samples. The first part focuses on pipetting techniques and dilution calculations, essential for quantitative microbiological analysis. The second part emphasizes counting bacteria using plate counts and turbidimetric methods, vital for estimating bacterial concentrations in liquids. This comprehensive assignment requires students to understand theoretical concepts, perform precise calculations, interpret experimental data, and develop graphical representations of bacterial growth patterns. Successful completion demonstrates mastery of laboratory techniques, data analysis, and critical thinking necessary for microbiological research and quality control applications.

Objectives of the first exercise – Briefly describe the objectives in your own words, referring to the lab manual or PPt.

The primary objective of this exercise is to develop proficiency in pipetting accurately and executing serial dilutions to quantify microorganisms or solutes in samples. It aims to reinforce understanding of how dilution factors influence concentration calculations and enhance skills in precise liquid measurement. Additionally, it seeks to familiarize students with proper pipette handling, reading meniscus correctly, and performing calculations to determine the amount of solutes in diluted samples, which are critical for reliable microbiological and biochemical analyses.

Definitions: Solute, Solvent, Dilution, Solution

Solute: The substance dissolved in a solvent to form a solution, usually present in a smaller amount. For example, salt dissolved in water.

Solvent: The substance in which the solute dissolves; typically a liquid such as water.

Dilution: The process of reducing the concentration of a solute in a solution by adding solvent.

Solution: A homogeneous mixture formed when a solute is uniformly dissolved in a solvent.

Part One: Pipetting and Dilutions

Question B. 1

For each set of dilutions illustrated below, calculate the amount of colored substance (diluted sample) in the last test tube of each set. Show your calculation steps and results.

Question B. 2.1

What is the “meniscus”?

The meniscus is the curved surface of a liquid in a graduated cylinder or pipette caused by surface tension. When measuring liquids, readings are taken at the bottom of the meniscus at eye level for accuracy.

Question B. 2.2

If you transfer 0.1 mL of a sample into 99.9 mL saline blank, what is the dilution factor? Show calculation steps.

Solution: The dilution factor is calculated as the total volume divided by the volume of the sample transferred:

Dilution Factor = (Volume of dilution tube) / (Volume transferred) = (0.1 mL + 99.9 mL) / 0.1 mL = 100 mL / 0.1 mL = 1000

Question B. 2.3

How much fluid is in the pipette below?

[Answer should specify the volume contained in the pipette, based on the specific pipette used, e.g., 1 mL, 0.5 mL.]

Questions B. 3.1 – B. 3.3

Determine the following for each plate: the number of colonies (CFUs) within 30–300, the dilution factor, and the volume of diluted sample plated. Then, calculate the bacteria concentration in the original sample using the appropriate formula.

Example: If 150 colonies are counted on a plate where 0.1 mL of a 10^-5 dilution was plated, then the original concentration is calculated as:

CFU/mL = (Number of colonies) / (Volume plated in mL) × (Inverse of dilution factor)

CFU/mL = 150 / 0.1 × 10^5 = 1.5 × 10^8 CFU/mL

Part Two: Counting (Enumeration) of Bacteria

Question 2. 1

State the objectives of this exercise in your own words, based on the lab manual.

The objective is to learn how to accurately count viable bacterial cells in a suspension using plate counts, understand the principles of turbidity measurement, and correlate optical density readings with bacterial concentration through standard curves.

Question 2. 2

What is viable plate count?

Viable plate count is a microbiological technique used to estimate the number of living, culturable bacteria in a sample by counting the colonies formed on an agar plate after incubation.

Question 2. 3

What do you use to determine the number of bacteria in suspension by the turbidimetric method?

An spectrophotometer or colorimeter measuring absorbance at specific wavelengths (typically 600 nm) is used to determine bacterial turbidity, which correlates with cell concentration.

Question 2. 4.1 – 2.4.6

[Insert data table from PPt]

Explain why a standard plate count is necessary when first performing turbidity measurements.

Standard plate counts establish a correlation between optical density (turbidity) and actual viable bacterial concentration, allowing for accurate interpretation of spectrophotometric data.

Can the same graph be used for other bacteria like Staphylococcus if it was created for E. coli?

No, different bacterial species grow at different rates and have different optical properties, so separate standard curves should be developed for each.

How is “transmission” different from “absorbance”?

Transmission refers to the amount of light passing through a sample, expressed as a percentage, while absorbance measures the amount of light absorbed by the sample, which is directly related to bacterial concentration.

Provide the formula for calculating bacterial numbers in 1 mL or 1 gram of original sample, including calculation steps.

CFU/mL = (Number of colonies) / (Volume plated in mL) × dilution factor

Question 2. 4.6

Using the above formula, calculate the bacterial count per mL in the original E. coli suspension tube.

[Insert calculation based on hypothetical data]

Question 2. 4.7

Create a standard curve using Excel by plotting absorbance (X-axis) against CFU/mL (Y-axis). Include the graph with axes labeled appropriately and a trendline fitted to the data.

This curve allows prediction of bacterial concentration from absorbance values in unknown samples.

Conclusion

This assignment integrates fundamental microbiological techniques such as pipetting, serial dilution, colony counting, and spectrophotometry. Mastery of these methods enhances accuracy and reliability in quantifying microbial populations, essential for research, clinical diagnostics, and industrial microbiology. Proper understanding of data interpretation, graphing, and the principles behind each method ensures valid results and meaningful insights into microbial behaviors and responses.

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