LAB 7: RESPIRATORY SYSTEM

RESPIRATORY SYSTEM: UPPER AND LOWER

THE RESPIRATORY SYSTEM Host Defense Mechanisms

Anatomical characteristics/mechanisms to prevent/fight infection:– nasal hairs – antibacterial enzymes (lysozyme)– convoluted passages – cilia and mucous lining of the trachea– mucous linings of the nasal turbinates – reflexes --coughing, sneezing, swallowing – Normal flora of nasopharynx and presence of normal flora

NORMAL MICROBIOTA OF THE RESPIRATORY SYSTEM

• UPPER respiratory tract :– Coagulase – negative staphylococci– Neisseria (Gram – negative cocci)– Corynebacterium spp. (Diphtheroids) (Gram – positive rods)– Anaerobic bacteria– Pathogens in low numbers (colonization vs. pathogenic) (S. aureus, Gram-negative rods (Enteric/Pseudomonas spp.)

• LOWER respiratory tract (trachea, bronchi, and bronchioles) is virtually free of microorganisms, mainly because of the efficient cleansing action of the ciliated epithelium

RESPIRATORY TRACT INFECTIONS

Bronchitis Pharyngitis

Epiglottitis Sinusitis

Pneumonia

PATHOGENS OF THE UPPER RESPIRATORY TRACT

THROAT:

•Streptococcus pyrogenes (Group A strep)•Viruses

SINUS:

•Moraxella catarrhalis (Gram – negative cocci)•Gram – negative rods•Staphylococcus aureus•Viruses

PATHOGENS OF THE LOWER RESPIRATORY TRACT

• Streptococcus pneumoniae• Hemophilus influenzae• Staphylococcus aureus• Moraxella catarrhalis (Gram – negative cocci)• Gram – negative rods (Klebsiella sp., Enterobacter sp., Serratia sp., E. coli, Pseudomonas sp.)• Fungi (Cryptococcus neoformans)• Viruses

The Respiratory System: Collecting Specimens & Diagnosis

• Upper respiratory tract: Mucus membrane (throat, sinus) is wiped with a special collection swab. – Culture for isolation, identification

and susceptibility testing of pathogens

• Lower respiratory tract: Sputum and or bronchial specimens are collected into a sterile container.

– Gram stain for white blood cells and microorganism and to check for the quality of the specimen

– Culture for isolation, identification and

susceptibility testing of pathogens.

MEDIASheep Blood agar:

•Enriched (contains 5% sheep blood with tryptic soy agar base)•Differential (used to detect hemolysis of microorganisms)

•Microorganisms produce toxins called hemolysins that can break down red blood cells causing a visual change in the media

IDENTIFICATION

•DIFFERENTIATION OF STAPHYLOCOCCI AND STREPTOCOCCI

– GRAM STAIN– CATALASE– HEMOLYSIS ON SHEEP BLOOD AGAR

GRAM STAIN

•STAPHYLOCOCCUS

•STREPTOCOCCUS

GRAM POSITIVE COCCI IN PAIRS ANDCLUSTERS

GRAM POSITIVE COCCI IN PAIRS ANDCHAINS

CATALASE TEST

Streptococci are Gram-positive, catalase negative (no bubbles)

Staphylococci are Gram-positive, catalase positive (bubbles)

Blood Agar - Hemolysins• Alpha-hemolysis – causes partial lysis of RBCs and

breakdown of hemoglobin which leads to a green color change in the agar.

• Beta-hemolysis – causes complete lysis of RBCs and breakdown of hemoglobin which leads to a colorless clearing in the agar.

• Gamma-hemolysis – an organism that does not cause hemolysis.

SHEEP BLOOD AGAR

BLOOD AGAR LAB PROCEDURE

BLOOD AGAR LAB PROCEDURE

Blood Agar Lab Procedure: RESULTS

GROUP A STREP (S. pyrogenes) – (causative agent of ‘Strep throat’)

Bacitracin (A) disk test

Group A beta strep is sensitive to bacitracin (zone of inhibition)

Other beta streps are resistant to bacitracin (no zone of inhibition)

Streptococcus pneumoniae• S. pneumoniae is an alpha

streptococci that is susceptible to the optochin disk (zone of inhibition)

• Most common cause of CAP

• Other alpha streptococci are resistant to optochin (no zone of inhibition)

Streptococcus pneumoniae• S. pneumoniae • Catalase negative• Gram – positive cocci in LANCET PAIRS• Frequently encapsulated

DICHOTOMOUS KEY FOR IDENTIFICATION OF STAPHYLOCOCCI AND STREPTOCOCCI

GRAM POSITIVECOCCI IN PAIRS, CHAINS OR CLUSTERS

CATALASE TEST CATALASE NEGATIVE = STREPTOCOCCI

CATALASE POSITIVE=STAPHYLOCOCCI

GAMMA HEMOLYSIS

HEMOLYSIS

BETA HEMOLYSIS – Bacitracin disk sensitive = S. pyrogenes (Beta hemolytic strep Group A)

ALPHA HEMOLYSIS – Optochin disk sensitive = S. pneumoniae

STAPHYLOCOCCI VS. STREPTOCOCCI

GRAM POSITIVE COCCIGRAM POSITIVE COCCIPAIRS AND CHAINS

PAIRS AND CHAINS

PAIRS AND CLUSTERSPAIRS AND CLUSTERS

CATALASECATALASENEGATIVENEGATIVE POSITIVEPOSITIVE

HEMOLYSIS

ALPHABETA

GAMMA

HEMOLYSIS

ALPHABETA

GAMMA

MSA PLATEMSA PLATEPOSITIVEPOSITIVE

NEGATIVENEGATIVE

Coagulase negative Staphylococci

Coagulase negative Staphylococci

S. aureusS. aureus