Microbiology Practical PDF

Sampling ❶ Identify ???  Blood culture bottle Steps of blood culture collection Uses ?? Diagnosis of bacteremia or septicemia Caused by streptococcus pyogenes & Proteus How you disinfect skin ?? ❷ By iodine then alcohol...

Sampling ❶ Identify ???  Blood culture bottle Steps of blood culture collection Uses ?? Diagnosis of bacteremia or septicemia Caused by streptococcus pyogenes & Proteus How you disinfect skin ?? ❷ By iodine then alcohol Amount of sample ?? 20 – 30 ml How is the sample stored? At room temperature (don’t refrigerate ) Sampling Identify ??? CSF sampling Uses ?? Diagnosis of meningitis or encephalitis Caused by pseudomonas & Proteus How you disinfect skin ?? alcohol, then betadine then alcohol Amount of sample ?? At least 5 ml How is the sample stored? 37°C or at room temperature (don’t refrigerate ) Site? lumbar spinal puncture and drain it slowly into clean sterile tubes. Sampling Identify ??? urine collection from catheter Uses ?? Diagnosis of Urinary tract infection Caused by E coli & Proteus How you disinfect skin ?? close to the outlet from patient body with alcohol (leave 20 sec.) Other method of urine sample collection ?? midstream clean catch and suprapubic aspiration How is the sample stored? in refrigerator (4°C ) Sampling Identify ??? Sputum sample Collection Uses ?? Diagnosis of Pneumonia (lower respiratory tract infection) Caused by Klebsiella & pseudomonas time of sample collection?? early in the morning before eating or using mouth wash How is the sample stored? < 2 hours at room temperature or refrigerate for up to 24 hour  Sampling Identify ??? Throat swab sample collection Uses ?? Diagnosis of upper respiratory tract infections Caused by Staphylococcus aureus What is the suitable sites for sample collection ?? posterior pharynx, tonsils, and inflamed areas What is unsuitable sites for sample collection ?? side of mouth & tongue  Sampling Identify ??? Pus swab from open wound Organism : pseudomonas & Staphylococcus aureus site : lesion's fresh border Identify ??? Sterile container. Uses ??? Urine / sputum sample Identify ??? sterile swab  Sampling Identify ??? Fungal specimens before examine this sample by LM must ??? using 10-30 % KOH solution to digest the keratin surrounding the fungi Skin scraping Nail pieces Hair sample  microscope Identify ??? Identify ??? Light microscope Electron microscope Mention 2 other types of microscope with uses. : higher resolving power. : To visualize the viruses Electron microscope & fluorescent microscope Mention 2 differences between it and fluorescent microscope : 1. ultraviolet rays used as a source of light 2. Certain dyes e.g. auramine O  Fungal identification Budding yeast like cells and pseudohyphae diagnosis (Medical important )??? diagnosis (Medical important )??? Dermatophyte fungal infection Candida fungal infection (superficial mycoses ) (opporunistic mycoses ) Morphological type. : Morphological type. : Filamentous fungi (molds) yeast like fungi technique : technique : Wet smear mixed with 10-30% KOH Wet smear.  Fungal identification diagnosis (Medical important )??? Cryptocccus neoformans with its Uses of this stains : capsule (opporunistic mycoses ) Histopathological diagnosis of fungal infection Morphological type. : yeast fungi (round or oval ) technique : Wet smear mixed with India ink stain ( -ve stain ) Other stain use for diagnoses of this organism : Mucicarmine stain  Fungal identification Identify ??? ??? Identify ??? ??? Yeast-like colonies of - Filamentous colonies on SDA. Candida on SDA A. Dermatophyte. B. Aspergillus fumigatus. C. Aspergillus niger. D. Zygomycetes  Fungal identification Diagnosis ??? Candida Diagnosis ??? Aspergillus Diagnosis ??? Stain ??? Gram +ve Stain ??? Dermatophytes Morphology ??? Lactophenol cotton blue Stain ??? oval budding yeast like cells Lactophenol cotton blue and pseudomycelium  LM Diagnosis ??? Motility of vibrio technique ??? Wet smear Step of gram stains : the specimen material is fixed by heating on a slide and stained by sequential exposure to : 1. Gram crystal violet. 2. iodine. 3. decolorizer (95 % ethyl alcohol). 4. counterstain (safranin). bacteria Identify ??? Gram +ve Cocci in cluster Example : Staphylococci. Mention another possible arrangements of this bacterial morphology.?? Chain & pairs What is the basis of the differential gram stain? Cell wall structure (peptidoglycan thickness) bacteria Identify ??? Gram - ve Cocci Example : -Neisseria , Haemophilus and Bordetella Mention the decolorizing agent used in this staining procedure..?? 95% ethyl alcohol bacteria Identify ??? Gram +ve Bacilli Example : Corynebacteria , Bacillus and Clostridium Mention 2 other bacterial morphologies.?? Spirochete & spirillum bacteria Identify ??? Gram –ve Bacilli Example : Enterobacteriaceae & Pseudomonas Mention the counterstain used in this staining procedure.?? Safranin  bacteria Identify ??? Identify ??? EM picture of virus Normal tissue culture cell line Mention 3 methods for direct detection of Mention types of cell lines: viruses : 1-Primary cell lines: primary rabbit kidney 1. Electron microscope (virus) 2-Semicontinuous cell lines: human 2. Direct immunofluorescent technique diploid cell lines 3. Sandwich ELISA (for Ag) 3- Continuous cell lines: Hela and HEP2 4. PCR (Real time PCR) cell lines  bacteria Identify ??? Identify ??? Immunoflourescence Real time PCR uses ??? identification and quantification of RNA or DNA (measurement of viral load)  bacteria Identify ??? Identify ??? syncytia or multinucleated giant cells tissue culture bottle and inclusion bodies Mention 2 methods of identification of Mention 2 methods for indirect detection of virus in tissue culture cells.: viruses : 1. Direct or indirect immunofluorescence 1. indirect immunofluorescence 2. Detection of virus specific genome (PCR) 2. Indirect ELISA ( for Ab ) 3. Neutralization tests  bacteria Identify ??? Cytopathic effect Mention its types.: rounding, shrinking, swelling of cells, formation of syncytia or multinucleated giant cells  bacteria Identify ??? ??? Neutralization test Identify ??? ??? Immunblotting western blot ‫ممكن يجي ارسم‬  bacteria Identify ??? ??? Haemagglutination test Uses ??? ??? Identification of viruses Ex :influenza virus Identify ??? Haemagglutination Identify ??? Identify ??? Haemagglutination inhibition test Haemagglutination inhibition  Serological tests diagnosis of a recent infection depends on ??? 1. Detection of IgM. 2. Rising titer of IgG 3. High stationary titer Advantages of Serological tests ??? ✓ Sensitive and specific (variable). ✓ Does not require organism to be isolated in pure culture. ✓ Can be done to non - culturable bacteria. ✓ Rapid and cost effective.  Serological tests 1 2 Identify ??? ??? positive negative 1. Positive agglutination test for Ab 2. Positive agglutination test for Ag Identify ??? ??? Latex agglutination uses ??? ??? Detect the soluble antigen or its antibody  Serological tests 1 2 Identify ??? ??? 1. direct immunofluorescence (DIF) ( uses: Ag detection ) 2. indirect immunofluorescence (IIF) ( uses: Ab detection ) Identify ??? ??? - IIF is more sensitive & magnification Coagglutination test than DIF uses ??? ??? Detect unknown Ag by coated Ab on Staph. Aureus protein A  Serological tests Identify ??? ??? ELIZA uses ??? ??? - Direct method for detection of unknown Ag in specimen - Indirect method for Ab detection in serum Principle ( ‫??? ??? ) ارسم‬ The concentration of color can be assessed by ??? ??? spectrophotometer Enzyme as ( peroxidase or alkaline phosphatase)  Serological tests Identify ??? ??? Lateral flow immunochromatographic assays uses ??? ??? - rapid detection of antigens (within 15 minutes)  Nucleic acid detection Identify ??? ??? Identify ??? ??? PCR machine Nucleic acid sequencing uses ??? ??? uses ??? ??? - Nucleic acid Detection - determination of pathogen Examples of molecular assays used: genome sequence - PCR for DNA genomes, reverse transcriptase-PCR for RNA genomes  Nucleic acid detection 1 2 The main types of culture media are: 1. Enriched 2. Anaerobic 3. Basal 4. Transport 5. Differential (selective) Identify ??? ??? 1. Nutrient broth (fluid) 2. Nutrient agar Type ??? Basal or ordinary media uses ??? - Growth of non fastidious bacteria e.g. staph.  Nucleic acid detection Identify ??? ??? Blood agar EX: streptococcus EX:streptococcus Type ??? pyogenes pneumoniae Enriched media uses ??? 1- Growth of fastidious eg. Streptococci 2. Discriminating organisms according to their hemolytic action on blood Preparation ??? ??? nutrient agar + 5-10 % sterile defibrinated blood  Nucleic acid detection Identify ??? ??? Identify ??? ??? Chocolate agar Mannitol Salt Agar Type ??? Type ??? Enriched media uses ??? Selective media uses ??? culture of Haemophilus spp and allows the growth of staphylococci Neisseria spp Selecting agent: NaCl (10-15 %), it allows the Preparation ??? ??? growth of Staphylococci blood + temperature is Sugar: Mannitol raised for 10 min  Nucleic acid detection Identify ??? ??? EX: EX: MacConkey's agar Type ??? 1. E.coli 1. Proteus Selective media 2. Klebsiella 2. Salmonella uses ??? 3. Shigella allows the growth of staphylococci Selecting agent: Bile salts Sugar: Lactose Nucleic acid detection