LAB 3 BIO 39 S24

docx

School

American River College *

*We aren’t endorsed by this school

Course

Subject

Biology

Date

Feb 20, 2024

Type

docx

Pages

Uploaded by gulalaim202

BIO 39 LAB 3 Microscopy and Simple Stain I. Microscopy Due to their small size, microbial cells must be viewed using a microscope. The compound light microscope is the principal microscope used to observe bacteria, yeast/molds, protozoa, and helminth eggs. The term light refers to the method by which light transmits the image to your eye. The term compound refers to the use of a two-lens system to magnify and focus the image of the microbial cell. The majority of microbial cells are colorless, making them difficult to see against a clear glass slide. To visualize the microbial cells, stains (dyes) are often used that bind to cellular structures of the microbial cell and provide contrast with the glass slide. Refer to Chapter 2 on Microscopy in the Microbiology Fundamentals textbook (pp 46-55) for additional information. A. Microbes seen by standard microscopy 1. 2. 3.  A. Microscope Function: 1. magnification: 1

2. resolution: B. Parts: write the function and location or description of the following 1. Condenser: 2. Iris Diaphragm: 3. Objective lens: a. 4X: b. 10X: c. 40X: d. 100X: 1. Refraction: 2. Immersion oil: 40X 100X 4. Ocular lens (10x): 5. Total magnification: 2

6. Course Focus: 7. Fine Focus: C. THE COMPOUND LIGHT MICROSCOPE RULES FOR MICROSCOPE USE Always observe the following rules for microscope use: 1. ALWAYS use both hands to collect and return the microscope to the storage cabinet 2. The lowest power objective (4X) should be in position both at the beginning and end of microscope use. No immersion oil should be on the microscope. 3. Focusing the Microscope: a. Always begin with the 4x objective lens. Use the course focus knob to bring the sample into general focus, then use the fine focus to bring the sample into sharp relief. Use the 4X lens to survey the entire field. Using the X-Y axis control, move the slide to a field (area) of interest b. Rotate the objective so that the 10X lens is over the slide. The lens will click into place. Use the fine focus knob only to adjust the focus. Move the slide to better center a field of interest. c. Repeat (b) with the 40X lens. d. To use immersion oil: 1. rotate the objective turret so it is in-between the 40X and 100X lenses (no lens in place). 2. add 1 drop of immersion oil directly to the sample on the slide 3. rotate the objective so that the 100x lens in immersed (touching) the immersion oil. If the 100x lens is not in the immersion, there will be no reduction in refraction and nothing will be seen. e. NEVER , UNDER ANY CIRCUMSTANCES, EVER ALLOW THE IMMERSION OIL TO COME INTO CONTACT WITH ANY OTHER LENS!!!!! The immersion oil will DESTROY the 40X, 10X, and 4X lenses. The 100X lens is specially designed to work with immersion oil. If immersion oil comes into contact with other lenses, immediately wipe it clean with lens paper. 4. Use only lens paper for cleaning lenses. 5. When finished, remove all oil from the microscope, including the 100X objective and Course/Fine focus knobs, and place the 4X objective in place. Cover the microscope and return it, in NUMERICAL ORDER, to the storage cabinet. Failure to properly care for the microscope, particularly in regards to immersion oil, will result in a point deduction from the total points from the student’s grade. See the BIO 39 syllabus for regarding use and care of microscopes. View prepared bacterial slide at 4X, 10X, 40X, and 100X 3

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

II. Bacterial Size, Shape, and Arrangement In properly diagnosing a patient for bacterial infection, the size, shape and arrangement of bacterial cells derived from a patient sample (wound, blood culture, urine, sputum) can provide very important information to the clinician as to the possible identity of the infectious agent. Therefore, it is essential that one know the parameters of bacterial dimensions and arrangements. A. Bacterial Size 1. Micrometer (  m): 2. Average ranges of bacteria cell: 3. Bacteria: 4. Eukaryotic microorganisms: 5. Viruses: B. Bacterial Shapes 1. Coccus ( pl . cocci): 2. Rod or bacillus ( pl . bacilli): 3. Spirochete :   C. Bacterial arrangements. Clinically significant bacteria may form characteristic cell arrangements (cell groupings) when obtained from a patient sample. Arrangements are an artifact of cell division. As one cell divides to become two, residual cellular materials can hold two or more cells together, thus producing an arrangement. 4

1. For coccus bacteria, the plane of division is responsible for the characteristic arrangement. a. division in one plane (Y axis) produces: 1. diplococci: Clinical species: Streptococcus pneumoniae : Neisseria meningitidis : Neisseria gonorrhoeae : 2. streptococci: Clinical species: Streptococcus pyogenes : Enterococcus faecalis/faecium : b. tetrad arrangement: c. sarcina arrangement: d. staphylococci: Clinical species: Staphylococcus aureus Coccus arrangements Cocci on skin and hair follicle 5

1. Bacillus: a single bacillus and streptobacilli: bacilli in chains 2. Spirochetes do not typically form any cell arrangements. **Noun versus adjective** The same term may be applied as both a noun and adjective. If the term is a noun, it will describe the genus or species name of a specific bacterium. Genus and species names are always italicized with the genus name being capitalized. For example Streptococcus pyogenes is the scientific name of a given bacterial species, Streptococcus being the genus name (noun). In contrast, the adjective streptococci (note the lower case spelling) is used to describe a group of cocci in chains, that may or may not be of the genus Streptococcus Determine size, shape and arrangement of normal flora by Simple stain A simple stain is a procedure in which cells can be observed where only a single dye is applied to the sample. The Simple stain procedure will allow you to distinguish prokaryotic cell based on morphology (size, shape, and arrangement) from other cell types. Bacterial species that inhabit the human mouth are able to attach to the epithelial cells of the cheek and gums as well as the teeth. The human epithelial cells are ~40X larger than the bacterial cells. Yeast cells are ~10X larger than bacteria. a. Slide preparation for simple stain YOU MUST WASH YOUR HANDS BEFORE YOU PROCEED 1. Obtain a glass slide. Using the black grease pen , draw a vertical line on the far-left side of the slide (orientation mark). Draw two circles on the slide. Mouth Yeast-bacteria 2. Using a sterile cotton swab, scrape the gum line and cheek of your mouth. Apply the collected material to the left circle. Discard the used swab in the cardboard biohazard box on your bench. 3. Obtain a pair of disposable gloves, put on your eyeware, and make sure your lab coat is completely closed. Using a sterile cotton swab, collect a sample from the yeast-bacteria mixture and roll the sample onto the right circle b. Air drying and fixation 1. Allow the slide to completely air dry. All fixed slides must be completely dry before proceeding with the stain procedure. a. purpose of air drying: to prevent cell lysis when exposed to fixing agent 2. Fixation of slide. All slides must be fixed before proceeding with the stain procedure. 6

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

a. purposes of fixation 1. 2. 3. c. procedure: be sure to have clothespin and paper towel available and place stain rack over the sink YOU MUST HAVE GLOVES, EYEWARE, AND LAB COAT TO PROCEED 1. Methanol fixation. Place the slide on the stain rack over the sink and flood the slide with methanol. Allow the slide to incubate for two minutes. Gently shake any excess methanol from the slide at the end of the fixation step. 2. Add a drop of crystal violet dye to the circle. Let sit for 1 minute. 3. Turn the tap of the water faucet so that the water is a trickle, collect the slide with the clothespin and thoroughly rinse the slide. BE VERY CAREFUL THAT THE WATER DOES NOT SPLATTER DYE ONTO YOU! 4. Lightly pat slide dry with a paper towel. Observe cells using 100X objective lens. 5. Draw the shape and compare the sizes for the bacterial and epithelial cells. The bacterial are often attached to the surface of the epithelial cells. Human Buccal epithelial cell (large cell) with attached bacteria (dark cells) Mouth (comments and drawing) ___________________________________ ___________________________________ ___________________________________ 7

Yeast-bacteria (comments and drawing) ___________________________________ ___________________________________ ___________________________________ d. Observe living protozoa and bacteria by wet mount Eukaryotic cells are substantially larger than bacteria and can be viewed as wet mounts that allow for the observation of motility. Procedure 1. Add one drop of the protozoa sample to a slide. It is helpful to collect the sample from the bottom of the container. Add a coverslip and view at 10X. Time-permitting: View slides of Giardia lamblia and Entamoeba prepared slides. D. AFB differential staining • 1. AFB differential stain: • Two dyes a. carbol fuchshin dye: b. methylene blue: stains all non-AFB cells blue (other bacteria, human cells, etc.) 2. 8 Mycobacterium tuberculosis

AFB Case Study A 31 year old male presented to the Emergency Department (ED) after experiencing gross hemoptysis (coughing with blood). The patient reports a 2 month history of productive cough, a 25 pound weight loss, night sweats, and fatigue. A chest X-ray (CXR) revealed bilateral cavitary infiltrates. The patient had a history of heavy alcohol and drug use with a long history of cigarette use and a chronic smoker’s cough. The patient reported no permanent residence. The patient was admitted and tested for bacterial pneumonia. Routine Gram stain from sputum (lower respiratory tract mucous) was negative. The patient is admitted to the hospital with a presumptive diagnosis of tuberculosis and placed in an AFB respiratory isolation room. Routine culture results were negative after 48 hours. AFB sputum cultures were collected on three consecutive mornings to ensure the best probability of detecting AFB. The second and third sputum samples produce 4+ AFB Ziehl-Neelsen staining (48 and 72 hours after admission). The patient was given isoniazid, rifampicin, and pyrazinamide. The patient experiences a decline in respiration with decreased blood oxygen saturation and is subsequently intubated for ventilation. The patient is placed on prophylactic Zosyn®. The patient remains intubated for the next 7 days. On day 8 the patient is extubated and placed on nasal cannula to improve oxygenation. On day 14 of hospitalization, the microbiology laboratory identifies M. tuberculosis from the patient’s AFB cultures. The patient was discharged on day 16 of hospitalization with a one-month supply of isoniazid, rifampicin, and pyrazinamide. Use the table below and answer the following questions. Answer the following questions: 1. What are the 7 signs and symptoms presented by the patient in the Emergency Department that are indicative of TB? 2. What is the significance of the chest X-ray results? 9

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

3. Are the Gram stain results consistent with an AFB/TB diagnosis? Explain your answer. 4. What is the importance of placing the patient in respiratory isolation? Would specific PPE be needed for the health care provider and patient be needed while the patient is in the Emergency Department? 5. What is the significance of direct microscopic examination of sputum sample in the AFB culture? 6. Explain how the classification of the sputum smear (slide 4+-0) is related to the infectiousness of the patient. E. Fever and White Blood Cell (WBC) Fever is defined as a temporary increase in the body's temperature in response primarily to infection from 98.6 o F (37 o C) as determined by vital signs assessment. Fever is any body temperature > 100.4 o F (> 38 o C) and the increase in body temperature is commensurate with severity of infection. example  body temperature of 101 o F (38.3 o C) indicates infection  body temperature of 107.5 o F (42 o C) indicates severe infection Increased body temperature has specific effects. Immune system processes are more efficient at higher body temperatures and certain pathogens replicate more slowly under higher body temperatures. High, prolonged fevers may cause neurological disruptions that may be permanent (>107 o F or 41.5 o C) and requires mitigation. White blood cells (WBCs) are the cellular components of the immune system. Elevated WBCs above healthy range are associated with disease and represent immune system activation. Infections are a major cause of elevated WBCs. The healthy range for WBCs is 4,500-11,000 10

WBC/  l and WBC levels >12,000 WBC/  l represent an elevated count (above normal value). Increased WBC count indicates severity of infection. example  16,000 WBC/  l = infection  37,000 WBC/  l = severe infection A 28-year-old patient involved in a house fire was brought to the Emergency Department (ED). The patient has sustained extensive third and fourth degree burns to the right leg, right hip, lower left leg, right lower arm, and right side of face. The patient has inhalation (smoke and heat) related damage to the bronchi and lungs. The TBSA is 40%. The patient was intubated due to respiratory damage. The patient is admitted to the Burn Center. Vital signs are assessed every four hours and WBC levels every eight hour. Evaluate the table below. 11 Test Healthy range 0500 1300 2100 Heart rate 60-100 bpm 80 99 121 Respiration rate 12-16 (20) bpm 14 17 23 Body temperature 98.6 o F (37 o C) 98.6 o F 100 o F 103.2 o F Blood Pressure 90/60-120/80 107/68 90/62 79/52 SaO 2 95-100% 99% 92% 88% WBC, total 4,500-11,000/ml 9,574 11,588 16,239

Pre-Lab 3 Name:______________________________________________ Lab Section__________ Read the following from the Microbiology Fundamentals: A Clinical Approach textbook pages 362 and 364, the text above on fever and white blood cells pp. 8-9, and Take-Home Assignment 1 to answer the following questions. The material will be used for questions on Lab Exam I . Questions 1. Define the term fever. What represents an elevated body temperature (fever)? Fever is an abnormally elevated body temperature, typically defined as a core temperature above 37.7°C (100°F). 2. Define the term pyrogens. (textbook reading) Pyrogens are substances that trigger fever by resetting the body's thermostat to a higher temperature. They can be exogenous, originating from outside the body (e.g., bacterial endotoxin), or endogenous, produced internally by immune cells (e.g., interleukin-1 and tumor necrosis factor). 3. How does fever correlate with severity of infection? Fever correlates with the severity of infection, with higher fevers often indicating more serious infections. Fevers above 104°F to 105°F may indicate a need for medical intervention to prevent tissue damage. 4. Cite three benefits of fever. (textbook reading) - Inhibiting the multiplication of temperature-sensitive microorganisms. - Interfering with the nutrition of bacteria by reducing the availability of iron. - Stimulating immune reactions and protective physiological processes, such as increasing metabolism, hematopoiesis, and phagocytosis. 5. How does elevated WBC number correlate with severity of infection? Elevated white blood cell (WBC) counts can correlate with the severity of infection. Infections often increase WBC production as the body mobilizes its immune defenses to 12

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

fight off pathogens. However, the specific correlation between WBC count and severity of infection may vary depending on the type and stage of infection. 6. Evaluate the three sets of data (0500, 1300, and 2100) Time of assessment a. Provide an explanation for the change in values from 0500 to 2100. Infection got more serious, and the numbers represent the immune system fighting. b. What does the elevated heart and respiration rates indicate? Increased heart rate and respiratory rate indicate that the body needs more oxygen. c. Provide a rationale as to why the is SaO 2 decreasing. SaO2 levels are falling as the body's temperature rises, causing hemoglobin to degrade. d. What does the increased WBC count suggest? Increased WBC levels indicate that the illness is worsening and that more WBC are required to combat it. 13 Test Healthy range 0500 1300 2100 Heart rate 60-100 bpm 80 99 121 Respiration rate 12-16 (20) bpm 14 17 23 Body temperature 98.6 o F (37 o C) 98.6 o F 100 o F 103.2 o F Blood Pressure 90/60-120/80 107/68 90/62 79/52 SaO 2 95-100% 99% 92% 88% WBC, total 4,500-11,000/  l 9,574 11,588 16,239

LAB 3 BIO 39 S24

Related Documents