تقارير الدروس العملية

Transcription

1 وزارة التعليم جامعة الباحة كلية العلوم الطبية التطبيقية قسم طب المختبرات تقارير الدروس العملية مقرر أحياء دقيقة إكلينيكية الدكتور : شائع بن صالح المالكي 5341 ه ه

2 Routine of Laboratory Diagnosis of Bacterial Infections in Clinical Samples Clinical Samples Commensals (normal bacteria) Examples of Possible bacterial Pathogen Culture Media Incubation conditions Temperature O2 and CO2 Blood Blood should be sterilized Staphylococcus epidermidis Staphylococcus saprophytics Streptococcus pyogenes Streptococcus agalactiae Streptococcus pneumoniae Heamophilus influenzae Neisseria meningitides Salmonella spp. Escherichia coli Pseudomonas aeruginosa Clostridia spp.. Listeria spp Candid albicans (Single cell Fungi) Mannitol salt agar MacConkyes agar Aerobic Anaerobic CSF Blood should be sterilized Neisseria meningitides Escherichia coli Pseudomonas aeruginosa Candid albicans (Single cell Fungi) Mannitol salt agar MacConkyes agar The optimum temperature of most pathogenic bacteria is 37c Urine Urine should be sterilized but sometimes, the skin normal flora can be seen. These, however, could be significant pathogens in some cases. Gram negative bacteria (Enterobacteriacea) are significant pathogens for urinary tract Escherichia coli Klebsilla spp. Proteus spp. Pseudomonas aeruginosa Serattia spp CLED agar MacConkeys agar also Staphylococcus spp. Candid albicans (Single cell Fungi) Stool Stools have mixtures of gram negative bacteria that live as normal flora in the intestinal tract. Therefore, selective media must be used Salmonella spp. Shigella spp. Vibrio cholera Brucella spp. Listeria spp. Bacillus cerus Selenite broth XLD agar Mannitol salt agar MacConkyes agar TCBS agar Sputum Blood should be sterilized. However, some mouth normal flora can be seen Mycobacterium tuberculosis (TB) Streptococcus pneumoniae Klebsiella pneumoniae

3 Throat Swabs some mouth normal flora can be seen Streptococcus pyogenes Streptococcus agalactiae Heamophilus influenzae Candid albicans (Single cell Fungi) Mannitol salt agar Wound Swabs some skin normal flora can Streptococcus pyogenes Escherichia coli Enterococci Pseudomonas aeruginosa Clostridia spp. Mannitol salt agar MacConkyes agar Vaginal and Urethral Swabs Urethra should be sterilized but the vagina may contain some normal flora that are important reduce the opportunity of infections with pathogens Neisseria gonorrhea And maybe other gram negative bacilli Staphylococcus spp Candid albicans (Single cell )Fungi MacConkyes agar

4 Identifications of some bacterial cultures on some media Bacteria Media Characteristics of colonies White or golden surrounded with beta hemolysis Chemical disks Photos Staphylococcus aureus White or golden Methicillin and Vancomycin Mannitol Salt Yellow Other Staphylococci Same media White colonies and no hemolysis Novobiocin Staph. Saprophytics (resistant) Staph. Epidermidis (sensitive)

5 Identifications of some bacterial cultures on some media Bacteria Media Characteristics of colonies Chemical disks Photos Sensitive to bacitracin Strept. pyogenes Small transparent gives complete hemolysis (Beta) Resistant to bacitracin Strept. agalactiae They need blood agar or chocolate agar Small transparent gives complete ))hemolysis (Beta CAMP test Positive Sterpt. pneumoniae Incomplete hemolysis (alpha) Sensitive to optochin Strept. viridans Incomplete hemolysis )(alpha Resistant to optochin

6 Identifications of some bacterial cultures on some media Bacteria Media Characteristics of colonies Photos Escherichia spp. MacConkeys Pink (lactose fermenters) Klebsiella spp. MacConkeys Mucoid pink (lactose )fermenters MacConkeys Pale (yellow) (non lactose fermenters) Proteus spp. Swarming Because of their high motility Pigmented (produce pigments) Pseudomonas spp. Pigmented (produce )green pigments Salmonella spp. MacConkeys Pale (non lactose fermenters) XLD agar pink colonies with black centers as result of producing H2S Shigella spp. MacConkeys Pale (non lactose fermenters) XLD agar pink colonies with black centers as result of producing H2S Vibrio cholera TCBS agar Yellow colonies as result of fermenting sucrose that is present in the media

7 Most of gram negative bacteria can grow on ordinary of bacterial media. It is needed however, to grow them in special media to select and identify them. Identifications of some bacterial cultures on some media Bacteria Media Characteristics of colonies Photos Mycobacterium tuberculosis Lowenstein-Jensen (LJ) The bacteria need long time (about 3-4 weeks) to be seen as colonies because its long generation time. Single bacteria need about hrs to divide into two cells. Therefore, diagnostic lab will go to direct stain (Zhile Nelson) as it is quicker and also safer as growing the bacteria may cause dangerous contamination. Advanced labs use PCR (genetic analysis). The bacteria can also be detected in blood using BACTEC system Neisseria meningitides This bacterium needs chocolate as it has X factor (Heamatin) facilitated. In the blood agar this is not free, so may be difficult to grow Neisseria gonorrhea This bacterium needs chocolate as it has X factor (Heamatin) facilitated. In the blood agar this is not free, so may be difficult to grow. There are special selective media to grow them i.e. Thayer-Martin agar New York City media Heamophilus spp. This bacterium needs chocolate as it has X factor (haemin or other porphyrins) and V factor (NAD, nicotinamide adenine dinucleotide) facilitated. In the blood agar this is not free, so may be difficult to grow. They can grow on media that is supported with these factors.

8 Biochemical Test The principle The Use Photo Catalase Breaking H202 into water and O2 that appears as bubbles Distinguish between staph(+) and strept(-) Coagulase An enzyme is produced by staph. aureus. It clots the plasma as it converts fibrinogen into fibrin Distinguish staph aureus from other staphylococci Oxidase Some bacteria able to oxidize the reagent tetra methyl para phenyl diamine hydrochloride and develop blue colour. Distinguish some bacteria such as Pseudomonas spp. and Neisseria spp Indole Tests the ability of organism to split indole from tryptophan H2S Urease It is the ability of the microbe to produce H2S on a medium, such as XLD, has sulphate Some bacteria produce urease, which splits urea into ammonia and carbon dioxide These tests are used to distinguish between gram negative bacteria (Enterobacteriacae), as they are very similar in morphology to each other. Although they are classified into major group Lactose Fermenters and Non Lactose Fermenters, they still needed to be more distinguished to achieve perfect diagnosis. biochemical tests are used to dist Black colour Gelatin Vogues Proskauer (VP) It tests the ability of the microbe to produce acetyl methyl carbinol from the fermentation of the glucose ONPG Citrate Identifies the ability of the microbe to synthese the enzyme beta-galactosidase. ONPG (Ortho-nitrophenyl-b-Dgalactopyranoside ) turns yellow in the presence of this enzyme. To test the ability of the microbe to utilize the citrate as a sole source of carbon Yellow Colour Note: These tests can be done separately. Nowadays these are in one ribbon which is called the API20E

9 Write the principle and procedure of the following staining techniques 1- Gram stain 2- Zhile-Nelson stain

10 Laboratory Diagnosis Line for some pathogenic Bacteria Negative cocci Negative bacilli Positive bacilli Positive cocci Catalase Oxidase and API20E Streptococci Negative Positive This provides a group of biochemical tests to help diagnosing them There are about 6 important types and can be distinguished on the basis of the differences of their antigenic structure using Lancefield Test Diplococci Streptococcus pneumoniae It is also sensitive to Optochine Serological test (antigenantibody reaction) can be used Staphylococci Coagulase Negative Positive Novobiocine Staphylococcus epidermidis Sensitive Staphylococcus saprophytics Resistant should be tested with methicilline to detect if it is resistant (MRSA) and also with vancomycine to detect if it is resistant (VRSA)