Diagnostic Microbiology I Lecture Notes PDF

# Lecture 9 ## Laboratory Diagnosis of Bacterial Infections (I) Dr. Rasha Mokhtar Elnagar M.B.B.C, MSc, PhD, MD Medical Microbiology and Immunology Associate Professor of Medical Microbiology & Immunology Consultant Microbiology & Immunology ## Laboratory Diagnosis of Bacterial Infections ### A-...

# Lecture 9 ## Laboratory Diagnosis of Bacterial Infections (I) Dr. Rasha Mokhtar Elnagar M.B.B.C, MSc, PhD, MD Medical Microbiology and Immunology Associate Professor of Medical Microbiology & Immunology Consultant Microbiology & Immunology ## Laboratory Diagnosis of Bacterial Infections ### A- Specimen collection and transport - General rules should be applied to all specimens. - The samples must be: 1. Taken before the start of antimicrobial therapy. 2. Representative of the infectious process: - Meningitis: CSF. - Respiratory Tract Infection (RTI): Throat and nasopharyngeal swab, sputum. - Intestinal infection: Vomitus, stool. - Urinary tract infection (UTI): Mid-stream urine. 3. Adequate volume. 4. Collected aseptically in an appropriate, sterile container. 5. Full request form (date, type of sample, department). 6. Transported rapidly to the laboratory. Use transport media if delay is anticipated. ### B- Bacterial identification * **Direct detection:** of the causative bacteria or their antigens or genetic material: 1. Microscopic examination. 2. Culture and Antibiotics sensitivity test. 3. Antigen detection 4. Molecular tests: PCR (detect genetic material) * **Indirect detection:** - Serology: detection of the specific antibodies. ## 1. Microscopic Examination ### Types of microscopes: - **Ordinary light microscope** (use ordinary light). - **Dark field microscope:** for detection of *Treponema pallidum*. - **Fluorescence microscope** (use UV): for detection of the *Mycobacteria* and the intracellular organisms e.g., *Chlamydia* & viruses. - **Electron microscope:** for viruses. ## Microscopical Techniques in Microbiology ### 1- Unstained (Wet mount preparation): - Fresh samples are used to demonstrate bacterial motility (e.g., *Vibrio cholerae*, *Salmonella*, *E. coli*, *Proteus*). ### 2- Stained: - Determine the staining reaction, shape and arrangement of bacteria. - **Gram stain.** - **Ziehl-Neelsen stain (Acid fast stain).** - **Special stains:** - Flagellar stain. - Capsular stain. ## Gram stain - The most important. - It classified bacteria into two major groups: - **Gram positive:** Appears Purple after Gram's stain. - **Gram negative:** Appears Red after Gram's stain. ### Gram Stain Steps 1. **Firstly:** **Fix** the smear of the specimen on the glass slide either by gentle heating or alcohol. 2. **Primary stain:** **Crystal violet** (methyl violet). 3. **Mordant:** **lodine**. 4. **Decolorization:** **Alcohol** (95% Ethanol). 5. **Counter stain:** **Safranin**. - **Examined with oil immersion lens (100x) after putting a drop of cedar wood oil on the stained smear.** ### Results: - **Gram Positive Bacteria:** appear **Purple** - **Gram Negative Bacteria:** appear **Red** ### Difference Due to Different Structure of Cell Wall of Gram Positive & Gram-negative Bacteria - **Gram positive:** **Thick** peptidoglycan layer (trap 1ry stain) - **Gram negative:** **Thin** peptidoglycan layer ## Ziehl-Neelsen Stain (Acid Fast) - Used to identify *Mycobacterium spp.* (high lipid and wax content in cell wall). - **Procedure:** 1. **Fix** the smear of the specimen on the glass slide either by heating or alcohol fixation. 2. **Primary stain:** **Carbol fuchsin.** (Heat gently until fumes appear). 3. **Decolorization:** Use **Sulphuric acid H2SO4** with **Alcohol 97%**. 4. **Counter-stain:** **Methylene blue**. - **Allow it to dry examine under oil immersion lens.** ### Result: - Acid fast bacteria e.g. *Mycobacterium tuberculosis* appear red. ## 2. Bacterial culture - Clinical samples are inoculated on different types of **media** under suitable **temperature** and **atmospheric** conditions. ### Classification of Media According to Consistency: - **Liquid** (broth). - **Solid or Semi solid** (agar plates; solidified by adding agar). ### Classification of Media According to Ingredients & Uses: - **A- Transport media:** - Maintain the viability of all organisms in the specimen until being transported to the laboratory for cultivation. - **Example:** Stuart transport medium, Amies medium, Carry & Blair medium. - **B- Culture Media:** - **1- Simple (Ordinary)medium:** - It contains the basic nutritive requirements. - **e.g.** *Nutrient agar* and *Nutrient broth* media. - **2- Enriched media:** - Support the growth of **Fastidious** bacteria. - **Enriched by adding:** - **Serum:** *Loffler's serum agar* - **Whole blood:** *Blood agar*, *Chocolate agar*. - **Blood agar** can be used as **indicator** or **differential media**, according to type of hemolysis produced by bacteria on blood agar: - β (Complete). - a (Partial). - y (No hemolysis). ## Case Study - A 45-year-old man presents to an emergency room complains of a 6-month history of night sweats, fever, and cough with the production of sputum. - He also states that he has lost about 30 Kg from his weight in the last year. - The initial clinical diagnosis was *Mycobacterium tuberculosis* infection. - Which of the following stain could help in diagnosis of *Mycobacterium tuberculosis*? - **A. Gram stain** - **B. Lactophenol cotton blue** - **C. Ziehl-Neelsen stain** - **D. India ink stain** ## Reference Lippincott Illustrated Microbiology 4th edition, Chapters: 4.