labs
Weekly Experiments in Microbiology Lab with Detailed Procedures
Week 1: Antimicrobial Susceptibility Testing
Objective: Determine the effectiveness of different antibiotics against specific bacterial pathogens (e.g., E. coli, Staphylococcus aureus).
Equipment:
Mueller-Hinton Agar plates: standardized medium for antimicrobial susceptibility testing.
Antibiotic-impregnated disks: contain known concentrations of antibiotics.
Inoculating loop: for transferring bacterial culture.
Incubator: set to 37°C for optimal bacterial growth.
Ruler or caliper: for measuring zones of inhibition.
Procedure:
Preparation of Bacterial Suspension:
Inoculate a single bacterial colony into a tube of sterile broth (e.g., nutrient broth) and incubate overnight at 37°C.
The next day, dilute the culture to achieve a 0.5 McFarland standard (equivalent to approximately 1.5 × 10^8 CFU/mL).
Inoculation of Agar Plates:
Using a sterile inoculating loop, dip it into the bacterial suspension and spread it uniformly across the surface of the Mueller-Hinton Agar plate to obtain a lawn of growth.
Let the plate sit for 15 minutes, allowing the bacteria to adhere.
Placing Antibiotic Disks:
Use sterile forceps to carefully place antibiotic disks on the inoculated agar, ensuring even spacing (approximately 2 cm apart).
Incubation:
Incubate the plates upside-down to prevent condensation on the agar surface for 24 hours at 37°C.
Measuring Zones of Inhibition:
After incubation, observe the plate for clear zones around the disks where bacterial growth is inhibited.
Use a ruler or caliper to measure the diameter of each inhibition zone in millimeters and record the results.
Week 2: Blood Smear Preparation and Staining
Objective: Prepare and stain peripheral blood smears to analyze cellular components and types in blood (erythrocytes, leukocytes, and platelets).
Equipment:
Glass microscope slides: for the smear.
Giemsa and May Grünwald stain solutions: for staining the blood components.
Microscope: for examining the stained smear.
Pipettes: for transferring blood sample.
Procedure:
Blood Collection:
Using a sterile lancet, obtain a drop of blood from a finger prick or collected sample tube.
Making the Smear:
Place a small drop of blood onto a clean microscope slide.
Use another glass slide at a 30-degree angle to touch the drop and gently spread it by moving the slide across the surface of the first slide.
Aim to create a thin, even smear that covers at least two-thirds of the slide.
Air Drying:
Allow the smear to air dry completely in a horizontal position without any drafts.
Fixation:
Once dry, fix the smear by dipping the slide into methanol for 3-5 minutes. This preserves the cells and enhances staining.
Staining:
Stain the dried smear with Giemsa solution for 5 minutes, then rinse with buffer or distilled water.
Counterstain with May Grünwald solution for another 10 minutes and rinse again thoroughly.
Microscopic Examination:
Allow the slide to dry, then examine under the microscope starting at lower magnifications (10x) before progressing to higher powers (100x) to identify cellular components.
Week 3: Gram Staining Procedure
Objective: Classify bacterial isolates into Gram-positive or Gram-negative based on cell wall characteristics.
Equipment:
Glass slides: for preparing the bacterial smears.
Gram stain reagents:
Crystal violet (primary stain)
Gram's iodine (mordant)
Ethanol or acetone (decolorizer)
Safranin (counterstain)
Microscope: for viewing stained slides.
Procedure:
Preparing Bacterial Smear:
Transfer a small amount of the bacterial colony to a slide and mix with a drop of sterile water to create a thin smear.
Air dry and fix with heat.
Staining Steps:
Cover the smear with crystal violet for 1 minute, then rinse gently with water.
Apply Gram's iodine for 1 minute, rinsing again afterwards.
Decolorize by pouring ethanol over the slide for 10-30 seconds until no color runs off, then quickly rinse with water.
Counterstain with safranin for 30 seconds, rinse, and dry.
Microscopic Observation:
Observe stained slides under the microscope, identifying the color of bacterial cells to determine classification: purple for Gram-positive and pink for Gram-negative.
Week 4: E-Test for Minimum Inhibitory Concentration (MIC)
Objective: Utilize the E-Test to find the MIC of antibiotics against specific bacteria.
Equipment:
E-test strips: containing a gradient of antibiotic concentrations.
Mueller-Hinton Agar plates: as the growth medium.
Incubator: set at 37°C.
Inoculating loop: for streaking bacteria.
Microscope: for observing bacterial growth.
Procedure:
Inoculation of Agar Plate:
Prepare a bacterial suspension similar to Week 1 and spread onto a Mueller-Hinton Agar plate to create a uniform lawn.
Applying E-Test Strips:
After inoculating, carefully apply E-Test strips to the surface of the agar using sterile forceps, ensuring they are in contact with the agar.
Incubation:
Incubate plates for 24 hours at 37°C in an inverted position.
MIC Assessment:
Examine the plate after incubation; measure where the growth intersects with the scale on the E-strip to determine the MIC value accurately.
Week 5: Catalase Test
Objective: Differentiate between catalase-positive and catalase-negative bacteria, which is critical for identifying species.
Equipment:
Hydrogen peroxide (H2O2): typically 3% solution.
Glass slides: for performing the test.
Inoculating loop: for transferring bacterial colonies.
Microscope: optional for observing colony details pre-test.
Procedure:
Preparation:
Use a sterile loop to pick a small amount of the bacterial colony from an agar plate.
Performing the Test:
Place the colony on a clean glass slide and add one drop of hydrogen peroxide directly on top of the bacterial material.
Observation:
Watch for the immediate formation of bubbles; a positive result (indicating catalase activity) is characterized by rapid bubbling due to oxygen release.
No bubbling indicates a negative result, suggesting the absence of catalase enzyme in the bacterium being tested.