Microbial Interactions with Humans

Microbial Interactions with Humans

Types of Interactions: Symbiosis
* Symbiotic Relationships

Overview of Human-Microbial Interactions
* Pathogens
* Pathogenicity
* Virulence
* Opportunistic Pathogen

Infection Versus Disease
* Infection
* Disease

Opportunistic Pathogens
* Don’t normally cause disease, but may under some circumstances
* 3 circumstances for gaining control/disease

Types of Interactions:
Normal Flora
* Normal Flora
* Factors that influence normal flora

Normal Flora
* Hundreds of different niches associated with human
* Some normal flora are pathogenic
* Resident versus transient flora
* Considered part of the first line of defense!
* Microbial antagonism
* Competitive exclusion

Portals of Entry
* Skin
* Mucous membranes

Preferred Portal
* Many microorganisms have to enter in a specific way and in a certain place to cause disease.
* Skin Portal

Skin
* Epidermis and Keratin
* Hair often deters microbial contact with skin
* Dermis and subcutaneous tissue
* Apocrine and sebaceous glands
* Eccrine glands (sweat)

Skin as a Barrier
Mucous Membranes
* Found in mouth, pharynx, esophagus, GI, respiratory, and urinary tracts
* Epithelial cells coated with protective glycoprotein layer (mucous)
* Less protection than skin
* Cilia and mucous produced by goblet cells

Mucous Membranes
* Respiratory tract Portal
* Respiratory Normal Flora
* Respiratory Barrier Mechanisms
* Gastrointestinal tract portal
* GI Normal Flora

Gastrointestinal Tract
* Large intestine
* GI Barrier
* Genitourinary tract Portal
* Genitourinary Tract Normal Flora
* Genitourinary Tract Barrier

LD50 and ID50
* LD50: Number of microbes in a dose that kill 50% of the organisms infected in a sample
* ID50: Number of microbes in a dose that causes disease in 50% of the organisms infected
* The higher the virulence the lower the ID50 or LD50

Microbial Virulence
Microbe Versus Host
* To cause disease a microbe must…
* Why it is difficult for microbes…
o Skin, antimicrobial sweat
Microorganisms and Mechanisms of Pathogenesis

Line of Defense
* First line: Skin and mucous membranes, normal flora
* Second line: phagocytes, inflammation, fever and antimicrobial substances
* Third line: (specific response) special lymphocytes (B and T cells) and antibodies

Step One: Adherence
* Specific adherence
* Pathogens have attachment structures
* Pathogens have attachment structures

Step 2: Invasion/Colonization
* Increase in numbers beyond the point of attachment.
* Three goals

Step 2: Invasion/Colonization
* Localized versus Systematic infections
* Bacteremia, viremia, toxemia
* Septicemia

Step 3: Cause Damage
* Virulence
* Three Ways to cause damage

Virulence Factors
* Usually help organism colonize and grow
* Coagulase
* Siderophores
* Collagenase
* Protease

Another Way to Classify Exotoxins
* Descriptive classifications
A-B toxin
* Cholera toxin (Vibrio cholera)—cholera

The Action of Chlorea Enterotoxin
More A-B toxin examples
Botulinum Toxin
Tetanus Toxin
Membrane Disrupting Toxins
Superantigens
Endotoxins
* Gram type negatives
* Part of outer portion of cell wall (outer membrane)
* Lipid A portion
* Exert effects when G- microbe lyses
* Same symptoms for different species of microbe
* No antitoxins produced by host
* Very stable—can’t destroy easily
* Rarely fatal
* Disseminated intravascular clotting
* General symptoms

Pyrogenic Response
* Macrophage ingestion
* Release of interleukin-1 in bloodstream
* Interleukin-1 to hypothalamus and production of prostaglandins
* Resetting of bodies thermostat

Susceptibility/Resistance of Host
* Species specificity
* Tissue specificity
* Age
* Stress
* Diet
* Pre-existing disease (Genetic and Infectious)
* Gender
* Behavior
* Weather?
* Your first line of defense—Review this

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Clinically Relevant Microbiology Starts at the Source

Clinically Relevant Microbiology Starts at the Source
By: Mike Costello, PhD, MT(ASCP)
ACL Laboratories
Mary Dikeman, MT (ASCP)
Affinity Health System

Program Objectives
* Emphasize that obtaining sensitive and specific microbiology results begins with the patient and not at the door of the microbiology laboratory.
* Accentuate the importance of proper collection and transport of specimens in both local and referral environments
* Stress the importance of timely communication between the Microbiology laboratory and those collecting specimens
* Describe common pitfalls in specimen collection and transport
* Discuss What rules or principles must be followed in order to collect microbiology specimens which will accurately reflect the pathogenesis of the microbiological agent. (Church D. The Seven Principles of Accurate Microbiology Specimen Collection. . Calgary Laboratory Services Microbiology Newsletter. Volume 6, 2005)

Introduction
The practice of sensitive, specific and cost effective clinical microbiology is intimately tied to the submission and proper handling of optimal specimens for analysis. Unfortunately, these aspects of clinical microbiology are not as critically controlled as our laboratory assays. It is our responsibility to educate and notify our healthcare colleagues when specimens arrive at the laboratory that will yield inferior results.

Quality assurance of specimen collection and transport is a never ending battle and requires long term commitment of your time and resources, but the end results are better patient care and a more rewarding experience for those of us who work in the microbiology laboratory.

Principle #1: The specimen must be collected with a minimum of contamination as close to
site of infection as possible

Urine Culture Contamination Rates

* Urine Culture contamination rates (>2 bacteria at >100,000 CFU) should be <20%
o CAP Q-Probe study (Valenstein P Meier F. Urine culture contamination: a College of American Pathologists Q-Probes study of contaminated urine cultures in 906 institutions. Arch Pathol Lab Med. 1998;122:123-129)..
+ 630 participants collected information of 155,037 urine culture specimens; 20.1% were considered contaminated (>2 organisms at >105 CFU)
+ The top 10% of institutions reported a rate of 5.6%. Bottom 10% of institutions reported a contamination rate of 36.8%
+ Males have a lower contamination rate than females (11.2% Vs. 22.8%)
+ ER departments had a contamination rate of 17.8%, sites adjacent to lab had rates of 19.5%, and other sites had rates of 22.1%

Blood Culture
* Two sets of blood cultures should be drawn. Number of sets positive correlates with true sepsis (except for coagulase negative Staph?) (Clin Microbiol. Rev 19:788-802, 2006)
* Catheter drawn blood cultures
o Catheter drawn blood cultures are equally likely to be truly positive (associated with sepsis), but more likely to be colonized (J Clin Microbiol 38:3393, 2001.)
+ One drawn through catheter and other though vein PPV 0f 96%
+ Both drawn from catheter PPV 0f 50%
+ Both drawn through vein PPV of 98%
o Study of positive coagulase negative Staphylococcus cultures and sepsis (Clin Infect Dis. 39:333, 2004.)

Blood Culture Contamination Rate
By Service Drawing Culture
What is an “Acceptable” Blood Culture Contamination Rate for Your Lab??
Blood Culture Contamination in Pediatric Patients
Young Children and Young Doctors
Inexperienced physician-young child
Inexperienced physician-older child
Experienced physician-younger child
Experienced physician-older child
Predicative Value of a Positive Result
False Positive
True Positive
Variable
Ped Infect Dis. 2006, 25:611-614.

Inexperienced Physicians = Interns and residents in 1st half of training
Experience Physicians = Residents in 2nd half of training and senior physicians
What is an “Acceptable” Blood Culture Contamination Rate for Your Lab??

What is an “acceptable” blood culture contamination rate*?
Berkeris LG, JA Toworek, MK Walsh, PN Valenstein. Trends in Blood Culture Contamination.
Arch Pathol Lab Med 129:1222-1294, 2005

Respiratory Cultures
* Community Acquired Pneumonia – Sputum rejection rate and culture correlation with gram stain
o 54% of all samples were judged to be of good quality.
o Presence of a (predominant morphology) PM on Gram stain was predictive of whether the sputum culture could demonstrate a pathologic organism. In the presence of a positive PM, 86% of cultures yielded a pathologic organism, while a positive culture was obtained in 19.5% of Gram stains without a predominant organism. S. pneumoniae was the most common infection, growing in 55.7% of positive sputum cultures.
o The sensitivity and specificity of finding Gram-positive diplococci for a positive culture of S. pneumoniae were 60% and 97.6%, respectively (Arch Intern Med. 2004;164:1725-1727, 1807-1811)
* Ventilator associated pneumonia (VAP) – appropriate specimen
o Blood cultures highly specific but not sensitive (positive in <10% of VAP)
o Quantitative cultures of lower respiratory tract specimens show a closer clinical correlation than sputum subcultures (Clinical Microbiol. Rev. 19:637-657, 2006.)

Viral Respiratory Cultures – Collect Sample From Site of Infection
How do you know that an adequate
Specimen was submitted for rapid
EIA assays???
Throat swabs are even worse!
Samples for Diagnosis of Viral Respiratory Infections
Lung biopsy
Bronchial alveolar
lavage/wash/brush
Nasopharygeal secretion
Nasopharygeal wash
Induced sputum
Nasopharygeal swab
Nasal wash


Throat swab (adenovirus only)
Saliva
Blood?
Sputum
DFA/EIA
OIA
Culture
LRTC*present
LRTC cells absent
Reagent Cost

Skin and Soft Tissue (Wound) Cultures
* Collect with steel (needle aspirate or scalpel)
* Discourage the use of swabs
* If infection NOT suspected, DON’T culture
* Get infected tissue or body fluid [ discourage swabs! ]
* -use something sharp ( syringe, scalpel, etc )
* -close doesn’t count
* *Don’t culture the surface / get deep infected sample*
* Remove needles / send capped syringe with aspirate
* Share specimen: Microbiology-Surgical Path-Cytology
* ** Label specimen and site accurately
* ** Give appropriate history
(Matkoski C. Sharp SE, Kiska DL. Evaluation of the Q Score and Q234 Systems for cost-effective and clinically relevant interpretation of wound cultures. J Clin Microbiol 2006;44:1869-1872)

Principle #2: A specimen must be collected at the optimal time(s) in order to recover the pathogen(s) of interest
Principle #3: A sufficient quantity of the specimen must be obtained to perform the requested tests
Blood Cultures
* Volume of blood drawn is the single most important factor influencing sensitivity. A single set for an adult blood culture consists of one aerobic and one anaerobic bottle. Optimally 10 mL of blood should be inoculated into each bottle. Volume of blood for a pediatric culture can be related to the infants weight
* Solitary blood cultures should be less than 5% (Arch Pathol Lab Med. 2001 125:1290-1294)
* If only enough blood can be drawn for one bottle, inoculate the aerobic bottle.
o 644 positive blood cultures, 59.8% from both bottles, 29.8% from aerobic bottle only and 10.4% from anaerobic bottle only (J Infect Chemother 9:227, 2003).
Pediatric Blood Cultures - Volume
Surgical Specimens (Other Shared Specimens)
TISSUE
FLUID
Specimen size of pea or larger
Divide
Anaerobic transport tube
Hold
upright,
uncap,
insert specimen and recap
Anaerobic
Culture
and stain

COLLECTION AND HANDLING OF OPERATING ROOM SPECIMENS FOR MICROBIOLOGY
Acceptable Specimens For Anaerobic Culture

Principle #4: Appropriate collection devices and specimen containers must be used to ensure recovery of all organisms
Recovery of Anaerobic Bacteria Placed in in Aerobic/Anaerobic Transport Media
CVP = Copan Vi-Pak Amies Agar Gel collection and transport swabs
SSS = Starplex StarSwab II,
PAC = BBL Port-A-Cult

How Does Transport Time Affect Yields?
J Clin Microbiol. 2001:39 377-380

Suggested Transport Media – General Comments

Principle #5: Collect all microbiology test samples prior to the institution of antibiotics
Principle #6: The specimen container must be properly labeled and sealed prior to transport
Principle #7: Minimize transport time or maximize transport media. There is always some loss of viability during transport
Minimize transport time and maximize use of transport media as much as possible
Environmentally Fragile Organisms
QA monitor??
Principle #8: Special handling/Collection instruction must be followed
* First, communicate with those that are doing collections.
* Collection instructions are written and available.
* Get involved with nursing orientation/education days and ask to have the instructions given out; poster board learning; quiz or competencies.
* Talk to providers when there are problems with specimen collection; they sometimes do not know they could do it better.


Principle #9: Improper specimen Collected for Ordered Test

Criteria For Rejection of Microbiological Specimens
* Criteria for rejection must be readily available and laboratory specific
* Unlabeled or improperly labeled specimen
* Prolonged storage or transport
* Improper or damaged container
* Specimen received in fixative
* Oropharyngeal contaminated sputum
* Duplicate specimens stools, sputum) within a 24 hour period. Exceptions cleared by the laboratory
* Specimens unsuitable for culture request (anaerobic culture from not acceptable source, urine from Foley catheter)
* Dry Swab
* 24-hr collection of urine or sputum for AFB or fungal culture
* Other criteria specific to your laboratory

Cultures That Should Include a Gram Stain
* CSF or sterile body fluid (cytospin)
* Eye
* Purulent discharge
* Sputum or transtracheal aspirate
* All surgical specimens
* Tissue
* Urethral exudates (male only, intracellular gonococcus))
* Vaginal specimens
* Wounds

Summary
* Publish specific rules for specimen collection
o There will be exceptions!
+ Make physician or healthcare provider aware of implications of culturing suboptimal specimens
* Communicate, communicate, communicate!
o Real time feedback
o Contact the health care worker who collected the suboptimal specimen

References

* Clinical Microbiology Procedures Handbook. 2nd Edition. . HD Isenberg ed. ASM. Cumitechs. ASM Press. Wash. DC.
* Manual of Clinical Microbiology, 9th Edition. ASM Press. Wash. DC. 2007.Miller MJ.
* A Guide To Specimen Management in Clinical Microbiology. ASM Press. Wash. DC. 1999.

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Specimen Collection and Laboratory Diagnosis of Lower Respiratory Infections

Specimen Collection and Laboratory Diagnosis of Lower Respiratory Infections

By:Mohammad Rahbar (PhD)
Department Of Microbiology Reference Laboratory of Iran

Anatomy of Respiratory Tract

“ The culture of lower respiratory specimens may result in more unnecessary microbiologic effort than any other type of specimen.”
Raymond C Bartlett

Lower Respiratory Tract Infections
Epidemiology
* Pneumonia is the sixth leading cause of death in US
* Increasing numbers of patients at risk
o Aging population
o Increase in patients with immunocompromising conditions
* Overtreatment has lead to resistance
o Multidrug resistant Streptococcus pneumoniae
o Resistance among hospital acquired pathogens such as Acinetobacter, Pseudomonas aeruginosa E.coli K.pneumonia (ESBLs) MRSA and others
* Major sections
o Clinical aspects of diseases of LRT
o Specimen collection
o Specimen processing
o Interpretation of bacterial cultures
o Most common pathogens
o Methods for implementing change
o Guidelines for frequency of testing
o Public health issues
o Reimbursement codes

Categories of Lower Respiratory Tract Infections
* Acute bronchitis
* Community acquired pneumonia
* Hospital acquired pneumonia
* Pneumonia in the immunocompromised host

Community Acquired Pneumonia Etiologic Agents
Community Acquired Pneumonia Diagnosis

Available Test Methodologies
* Sputum Gram stain and culture
* Blood cultures
* Serologic studies
* Antigen detection tests
* Nucleic acid amplification tests

Sputum Gram Stain and Culture
Proponents
* Demonstration of predominant morphotype on Gram stain guides therapy
* Accuracy is good when strict criteria are used
* Cheap, so why not?

Antagonists
* Poor specimen collection
* Intralaboratory variability (Gram stain interpretation)
* Low sensitivity and specificity
* Empiric treatment guidelines
* Not cost effective

Sputum Collection
* Proper patient instruction
o Food should not have been ingested for 1-2 h prior to expectoration
o The mouth should be rinsed with saline or water
o Patient should breathe and cough deeply
o Patient should expectorate into a sterile container
* Transport container immediately to lab
* Perform Gram stain and plant specimen as soon as possible

Sputum collection
* Sputum of less than 2ml should not be processed unless obviously purulent
* Only 1 sputum per 24hr .submitted
* Some scoring system should be used to reject specimen that re oral contamination.
* Transportation in <2 hr is recommended with refrigeration if delays anticipated.
* Handle all samples using universal precautions.
* Perform Gram stain and plant specimen as soon as possible

Induced sputum
Patients who are unable to produce sputum may be assisted by respiratory therapy technician. Aerosol induced specimen are collected by allowing the patient to breath aerosolized droplets of a solution of 15% sodium chloride and 10% glycerin for approximately 10 minute . obtaining such specimen may avoid the need for a more invasive procedures ,such as bronchoscopy or needle aspiration, in many cases.

Gastric aspiration
* The gastric aspiration is used exclusively for isolation of acid-fast bacilli and may be collected from patients who are unable to produce sputum, particularly young children. The relative resistance of mycobacteria allows them to remain viable for a short period. Gastric lavage must be delivered to the lab immediately so that the acidity can be neutralized. Specimen can be first neutralized and then transported if immediate delivery is not possible.

Sputum Gram Stain Unacceptable
Sputum Gram Stain Good Quality
Good quality specimens

* Quantify number and types of inflammatory cells
* Note presence of bronchial epithelial cells
* Concentrate on areas with WBCs when looking for organisms
* Determine if there is a predominant organism (> 10 per oil immersion field)
o Semiquantitate and report organism with descriptive
o If no predominant organism is present, report “mixed gram positive and gram negative flora”

Utility of the Gram Stain in Diagnosis of Pneumonia
Roson, B, et. al. 2000. Clin Infect Dis 31:869-74.

* Prospective study
* Non immunocompromised patients hospitalized with CAP
* 1,000 bed hospital in Spain
* ER physicians instructed on sputum collection for Gram stain and culture
* Sputum collected under supervision of nurse or resident
* Sputum collected under supervision of nurse or resident
o Samples were processed immediately
o Screened for epithelial cells
o Screened for predominant morphotype (> 75% of the organisms seen)
o Sputum planted to blood agar, chocolate agar and MacConkey agar
* Strictly defined clinical and diagnostic parameters

Utility of the Gram Stain in Diagnosis of Pneumonia
Roson, B, et. al. 2000. Clin Infect Dis 31:869-74

Results
* 190/533 (35.6%) patients had no sputum sample submitted (these patients were included in the calculations)
* 133/533 (25%) patients had a poor quality specimen
* 210/533 (39.4%) patients had a good quality specimen
* Overall sensitivity and specificity for pneumococcal pneumonia: 57% and 97%
* Overall sensitivity and specificity for H. influenzae pneumonia: 82 % and 99%
* Gram stain gave presumptive diagnosis in 80% of patients who had a good specimen submitted
* > 95% of patients in whom a predominant morphotype was seen on Gram stain received monotherapy

Gram Stain Reports
* Be as descriptive as possible
o Moderate neutrophils
o Moderate Gram positive diplococci suggestive of Streptococcus pneumoniae
o Few bacteria suggestive of oral flora
* Keep report short—avoid line listing of all morphotypes present

Sputum and Endotracheal Suction Culture Evaluation
* Identify and perform susceptibility testing on 2-3 potential pathogens seen as predominant on Gram stain
* Alpha strep—rule out S. pneumoniae
* Yeast—rule out Cryptococcus neoformans only
* S. aureus, Gram negative bacilli
o < normal flora, quantify and limit ID; no susceptibility
o Add comment that organism not predominant on stain
* ID mould, Mycobacteria or Nocardia spp.

IDSA Practice Guidelines
Diagnostic Tests for CAP
* Outpatients
o Empiric therapy with a macrolide, doxycycline, or a fluoroquinolone
* Hospitalized patients with CAP
o Gram stain and culture of sputum
o 2 pretreatment blood cultures
o Studies for Mtb, Legionella in select patients
Bartlett JG. 2000. Clin Infect Dis 31:347-82.
* Rationale
o To improve patient care
o Advance knowledge of epidemiologically important organisms
o Prevent antibiotic abuse
o Reduce antibiotic expense
Bartlett JG. 2000. Clin Infect Dis 31:347-82.

ATS Guidelines Diagnostic Tests for CAP
* Empiric therapy for outpatients
o Macrolide or tetracycline
* Hospitalized patients with CAP
o 2 sets of pre-treatment blood cultures
o Pleural fluid Gram stain/culture when appropriate
o Studies for Legionella, Mtb, fungi in select patients
o Sputum Gram stain/culture only if resistant or unusual pathogen is suspected
o Avoid extensive testing
ATS. 2001. Am J Respir Crit Care Med 163: 1730-1754.

Hospital Acquired Pneumonia
* Most frequent nosocomial infection (30-33% of cases) among combined medical surgical intensive care units
* 83% are ventilator associated
* Etiologic agents Frequency (%)
o Gram positive cocci
+ S. aureus 17
+ S. pneumoniae 2-20

AGENTS OF HAP
* Aerobic gram-neg bacilli 60
o Pseudomonas aeruginosa
o Enterobacter sp.
o Klebsiella pneumoniae
o Acinetobacter
o Legionella
o Anaerobes 10-20
o Fungi 0-10
Modified from: Carroll KC. 2002. J Clin Microbiol 40: 3115-3120.

Hospital Acquired Pneumonia Diagnosis

* American College of Chest Physicians: Clinical findings are not sufficient for definitive diagnosis
* Qualitative culture or endotracheal sputum has poor predictive value
* Bronchoscopy is recommended by many pulmonologists
o Bronchial brushings
o Bronchial washes
o Protected specimen brushing
o Bronchoalveolar lavage specimens (BAL)
o Transbronchial biopsy

Respiratory Specimens
* Protected Brush Specimen
o To procure uncontaminated lower airway secretions
o Brush within 2 catheters
* Bronchoalveolar Lavage (BAL)
o Samples large area of the lung
o Performed using a bronchoscope
o 100 to 250 ml of saline injected
o Injected saline along with secretions is collected by aspiration
* Transthoracic Aspiration
o Involves percutaneous introduction of a needle directly into the infiltrate

Bronchoalveolar Lavage (BAL) Specimen Acceptability
* Microscopic examination of Gram-stained smear
o Acceptable
+ <1% of cells present are squamous epithelial cells
o Unacceptable
+ >1% of cells present are squamous epithelial cells
Thorpe JE et. al. 1987. Bronchoalveolar lavage for diagnosing acute bacterial
pneumonia. J. Infect. Dis. 155:855-861

Processing Bronchoscopy Specimens
* Bronchoscopy brush protected
o Aerobic bacterial culture and Gram stain
o Anaerobic bacterial culture
o Limited volume
* Bronchoscopy brush, unprotected
o No anaerobic culture
o Limited volume
* Bronchial washings
o Useful only for pneumonia caused by strict pathogens
o Reasonable requests: Mtb, Fungi, Legionella, Pneumocystis
* Bronchoalveolar lavage
o No anaerobe culture
o Amenable to extensive testing for all opportunistic pathogens

Interpretation of Quantitative PSB/BAL
* Dilution Method
o Quantify each morphotype present and express as CFU/ml
* Calibrated Loop Method
o Quantify each morphotype present and express as log10 colony count ranges
* Thresholds for significance
o PSB > 103 CFU/ml
o BAL > 104 CFU/ml

Bronchoscopy Samples Quantitative Methods
Routine culture
* Most of the commonly sought etiologic agents of lower respiratory tract infection will isolated on routinely used media : 5% sheep blood agar ,MacConkey agar for isolation and differentiation of gram-negative bacilli ,and chocolate agar for Neisseria spp and Haemophilus
* Because of contaminating oral flora ,sputum specimens ,specimens obtained by bronchial washing, and lavage trachestomy, or endotracheal tube aspirates are not inoculated to enriched broth or incubated anaerobically. Only specimens obtained by percutaneous aspiration (including transtracheal aspiration )and by protected bronchial brush are suitable for anaerobic culture: he latter must be done quantitatively for proper interpretation.
* Transtracheal and percutaneous lung aspiration material may be inoculated to enriched thioglycollate ,as well as to solid media. For suspected cases of legionnaires disease buffered charcoal –yeast extract (BCYE) agar and selective BCYE are inoculated.

* Sputum specimens from patients known to have cystic fibrosis should be inoculated to selective agar ,such as manitol salt agar for recovery of S .aureus and selective horse blood-bacitracin ,incubated anaerobically and aerobically ,for recovery of H,influenzae that may be obscured by the mucoid P,aeroginosa on routine media. The use of selective medium for B.cepacia ,such as PC or OFPBL agar ,is also recommended

Immunocompromised Patients
Suggested BAL Protocol
* Aerobic Gram stain quantitative bacterial culture
* Fungal stain and culture
* Mycobacterial stain and culture
* Viral culture/Respiratory DFA
* Pneumocystis DFA
* Legionella culture

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Obtaining Specimens for Microbiological Evaluation

Obtaining Specimens for Microbiological Evaluation

Bacteremia I
* Most bacteremias are intermittent
* One blood culture is rarely sufficient
o Staphylococcus epidermidis
+ Frequent contaminant
+ Commonest cause of PVE
* Two blood cultures usually sufficient
o Three or four if suspect likely contaminant
o Antibiotic therapy

Blood Cultures - Volume
The magnitude of bacteremia may be low (<1cfu/ml)

Higher volumes have higher yield
Blood Cultures - Lab Aspects
* Additives (SPS, resins) increase yield
* Aerobic and anaerobic bottle = one blood culture
* Five days incubation sufficient
o Exception: Brucella, Histoplasma, Mycobacterium, Bartonella, Legionella
* Automated Systems detect CO2
o Subculture detected bottles

Aerobic/Anaerobic Blood Culture Bottles
AFB Blood Culture Bottle
Obtaining Blood Culture
* Locate the vein
* Prep kit
o Alcohol 5 sec. Dry 30-60 sec
o Tincture of Iodine-center to periphery. Dry 45-60 sec
* Remove caps, clean with alcohol
* Put on gloves
* Without palpating, draw 20 ml and put 10 in anaerobic and 10 in aerobic bottle
* Dispose of syringe in sharps container
* Label bottles and send to lab

Blood Culture Prep Kit
Sputum Culture Reliability
* Expectorated unreliable because of contamination
o Reliability  if physician observes
* Laboratory reliability screen
o > 25 PMN’s, < 10 oral squamous cells per hpf

Sputum Container
Sputum
* Gram stain
o Useful for immediate therapy
o May be more reliable than culture
+ Many PMN’s with single bacterial morphology
* AFB - first morning specimen
* Pneumocystis carinii - induced specimen

Nasal Cultures
* Virus
o Use wire swab
o Place in nose 1-3 cm, rotate, 10-15 sec
o Obtain viral transport medium from lab
* Bacterial
o Culturette with rigid or wire swab
o Suspect pertussis - special media

Wire Swab
Throat Cultures
* For Group A strept, diphtheria, gonorrhea
* Tongue blade - visualize pharynx and tonsils
* Rub swab over tonsils and pharynx
o INCLUDE ANY EXUDATE
* Insert into holder, crush vial

Swabs for Bacterial (red) and Viral (green) Cultures

Cerebrospinal Fluid
* Use sterile technique
* First or second tube to Microbiology
* Studies
o Gram stain - one drop cloudy fluid or sediment
o Aerobic culture - 1.0 ml
o Viral culture - 1.0 ml
o AFB or fungal culture - up to 10 ml

Wounds: General Principles
* Closed space infections provide reliable specimens
* Open wounds heavily contaminated
o May quantitate
* May obtain culture by aspirating advancing border
* Culture skin, soft tissue or wound abscesses for anaerobic and aerobic organisms
o Transport in capped syringe or special tube

Wound Culture
* Closed space abscesses
o Decontaminate skin
o Insert needle and aspirate or aspirate pus after incision
* Open wound
o Remove superficial exudate
o Aspirate through margin or swab (least reliable)
* Transport
o Capped syringe or anaerobic transport tube
o Rapidly to lab

Urine - General
* Collection method must avoid contamination
o Clean catch, midstream voided
o Catheterized urine
o Suprapubic aspiration
* Cultures performed quantitatively
o Less than 10,000 per ml suggest contamination

Clean Catch, Midstream Urine
* Cleanse periurethral area with soap and water
* Pass initial urine into toilet, then collect specimen in cup
* Instructions to patient are critical

Instructions for Patient
* Remove underpants completely so they will not get soiled.
* Sit comfortably on the seat, but do not leave your knees in front of you. Instead swing one knee to the side as far as you can.
* Spread yourself with one hand, and continue to hold yourself spread while you clean and collect the specimen.
* Wash—Be sure you wash well and rinse well before you collect your urine sample. Wash only the area from which you pass urine. You do not have to wash hard, but wash slowly. Be sure to wipe from the front of your body towards the back. Wash between the folds of skin as carefully as you can.
* Do not put sponges in the toilet. Put them back in the plate.
* Rinse—After you have washed with each soap pad, rinse with each moistened pad with the same front to back motion. Do not use any pad more than once.
* Hold cup by the outside and pass your urine into the cup. If you touch the inside of the cup or drop it on the floor, ask the nurse to give you a new one.

Catheterized Urine
* Cleanse periurethral area with soap and water
* DO NOT RECONTAMINATE
* Insert catheter into bladder
o Discard initial urine
o Collect specimen in sterile cup
* Chronic indwelling Foley catheter
o Clamp tubing below junction (or port)
o Disinfect with alcohol
o Insert needle (on syringe) through port or catheter wall and aspirate

Suprapubic Aspiration
* BE CERTAIN BLADDER IS FULL - PALPATE OR PERCUSS
* Prep skin with alcohol or iodine
* Anesthetize with lidocaine
* Introduce needle 2.0 cm above symphysis
* Aspirate 20 ml for culture

Suprapubic Aspiration
Wire Swab

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Surgical Preparation and Instrument Care

Surgical Preparation and Instrument Care

Sanitation, Disinfection and Sterilization

Learning Outcomes
After this section is completed you should be able to:
List the classes of pathogenic organisms in order of their resistance to destruction
Differentiate between sanitation, disinfection and sterilization
List the different ways that microbial control methods destroy or inhibit pathogenic organisms

After this section is completed you should be able to:
List the five categories of physical methods of microbial control
Name and describe the physical methods of microbial control
Identify the level of microbial control achieved with each of the physical methods
State an example of the application of each of the physical methods of microbial control

List the properties of the “ideal chemical agent” for microbial control
Name and describe the classes of microbial control chemicals
Identify the level of microbial control achieved by the chemical classes

List the advantages and disadvantages of the autoclave in animal care facilities
Explain the function of the autoclave
Compare and contrast the different autoclaves

Describe the preparation of each of the following for processing in the autoclave: linen packs, pouch packs, hard goods, liquids and contaminated objects
List the guidelines for loading the autoclave chamber
Compare the three different autoclave cycles

List and define the five methods of quality control for sterilization
List and define the two methods of quality control for disinfection

Section Outline
Levels of microbial resistance
Degrees of microbial control
How microbial control methods work
Methods of microbial control
Autoclave
Quality control for sterilization and disinfection

Overarching Principle
The objective in sanitation, sterilization and disinfection is to control microorganisms, or pathogens, in the environment, thus protecting patients and staff from contamination and disease, and thereby promoting optimal healing and wellness.

The Ever Present Danger
Improper application of the methods of sanitation, sterilization and disinfection can lead to microbial resistance and increase the risk of nosocomial infection

Levels of Microbial Resistance
Pathogens
Microorganisms that cause disease
Viruses
Bacteria
Fungi
Protozoan
Prions
Different classes of pathogens vary in their resistance to destruction by chemical methods

Protozoan Cysts
Bacterial Spores
Non-enveloped virus
TB organisms
Enveloped viruses
Fungi
Vegetative bacteria


Most Resistant
Least Resistant
Levels of Microbial Resistance
Microbial control
Is achieved by using methods of sanitation, disinfection and sterilization
Microbial control
Done to a degree that is practical, efficient and cost effective

Levels of Microbial Resistance
Sterility is used only when necessary

In many situations sanitation and disinfection create acceptable levels of microbial control

Degrees of Microbial Control
Sterilization is the elimination of all life from an object
Complete microbial control
Asepsis is a condition in which no living organisms are present
Free of infection or infectious material

Degrees of Microbial Control
Sanitation: The state of being clean and conducive to health.

Disinfection: To cleanse so as to destroy or prevent the growth of disease-carrying microorganisms

Degrees of Microbial Control
Disinfection, sanitation and cleaning remove most microorganisms
Most disinfectants are microbiocidal
Microbes are killed
Some disinfectants are bacteriostatic
Microbial growth is inhibited

Degrees of Microbial Control
Disinfectants can be classified according to their spectrum of activity
Bacteriocidal
Bacteriostatic
Sporocidal
Virucidal
Fungicidal

How Microbial Control Methods Work
Mode of Action
Different physical and chemical methods destroy or inhibit microbes in several ways
Damage cell walls or membranes
Interfere with cell enzyme activity or metabolism
Destroy microbial cell contents through oxidation, hydrolysis, reduction, coagulation, protein denaturation or the formation of salts

Efficacy of Microbial Control
The effectiveness of all microbial control methods depends on the following factors:
Time
Most methods have minimum effective exposure times
Temperature
Most methods are more effective as temperature increases

Efficacy of Microbial Control
The effectiveness of all microbial control methods depends on the following factors:
Concentration and Preparation
Chemical methods require appropriate concentrations of agent
Disinfectants may be adversely affected by mixing with other chemicals
Organisms
Type, number and stage of growth of target organisms

Efficacy of Microbial Control
The effectiveness of all microbial control methods depends on the following factors:
Surface
Physical and chemical properties of the surface to be treated may interfere with the method’s activity
Some surfaces are damaged by some methods

Efficacy of Microbial Control
The effectiveness of all microbial control methods depends on the following factors:
Organic debris or other soils
Will dilute, render ineffective or interfere with many control methods
Method of application
Items may be sprayed, swabbed or immersed in disinfectants
Cotton and some synthetic materials may reduce chemical activity

Methods of Microbial Control
Physical Methods
Chemical Methods

Physical Methods
Dry Heat
Oxidation
Moist Heat
Denatures microbial protein
Radiation
Damages cell enzyme systems and DNA
Filtration
Traps organisms that are too large to pass through the filter
Ultrasonic Vibration
Coagulates proteins and damages cell walls

Dry Heat
Incineration
Material or object is exposed to a hot fire
Object must become red hot as in the inoculation loops used in microbiology
Used to dispose of tissue or carcasses
Efficacy: complete sterilization

Dry Heat
Hot Air Oven
Sterility requires 1 hour of exposure @ 170° C(340° F)
Powders and non-aqueous liquids like paraffin or Vaseline
Used in some animal care facilities and useful in domestic applications (e.g. the kitchen oven)
Efficacy: complete sterilization

Dry Heat
Drying
Most organisms require humidity to survive and grow
More commonly used to prevent spoiling and preserve foodstuffs (e.g. raisins)
Efficacy: incomplete sterilization

Moist Heat
Hot Water
Used to clean and sanitize surfaces
Addition of detergents increases efficacy by emulsifying oils and suspending soils so they are rinsed away
Efficacy: incomplete sterilization

Moist Heat
Boiling
Requires 3 hours of boiling to achieve complete sterilization
Boiling for 10 minutes will destroy vegetative bacteria and viruses but not spores
Addition of 2% calcium carbonate or sodium carbonate will inhibit rust and increase efficacy
Useful for field work
Efficacy: may be complete sterilization

Moist Heat
Steam
Similar to boiling because the temperature is the same
Exposure to steam for 90 minutes kills vegetative bacteria but not spores
Efficacy: incomplete sterilization

Moist Heat
Steam under pressure
Pressure increases the boiling point such that the temperature of the water becomes much higher that 100° C (212° F)
The autoclave utilizes steam under pressure to achieve sterilization
This is the most efficient and inexpensive method of sterilization for routine use
Efficacy: complete sterilization

Radiation
Ultraviolet (UV)
Low energy UV radiation is a sterilant when items are placed at a close range
UV radiation has no penetrating ability
Used to sterilize rooms
Very irritating to eyes
Efficacy: may be complete sterilization

Radiation
Gamma radiation
Ionizing radiation produced from a Cobalt 60 source
Good penetrating ability in solids and liquids
Used extensively in commercial preparation of pharmaceuticals, biological products and disposable plastics
Efficacy: complete sterilization

Filtration
Fluid filtration
Forced through a filter with either positive or negative pressure
Filter is most commonly a synthetic screen filter with micropore openings
Used to sterilize culture media, buffers and pharmaceuticals
Pore size of 0.45µm removes most bacteria
Microplasmas and viruses require 0.01µm to 0.1µm
May be used in conjunction with a pre-filter
Efficacy: can be complete sterilization

Filtration
Air filtration
Examples of usage: surgical masks, laboratory animal cages and air duct filters
Fibrous filters made of various paper products are effective for removing particles from air
Efficacy is influenced by air velocity, relative humidity and electrostatic charge
Efficacy: can be complete sterilization

Filtration
Air filtration
HEPA: high efficiency particle absorption filters are 99.97% to 99.997% effective in removing particles with diameters greater that 0.3µm

Filtration
Air filtration
Surgical masks
Designed to protect the patient from the surgeon, not the surgeon from the patient
Special masks are available that are designed to protect personnel from animal pathogens
Masks must fit snugly, stay dry and be changed every 3 to 4 hours to remain effective

Ultrasonic Vibration
Cavitation
High frequency sound waves passed through a solution create thousands of cavitation “bubbles”
Bubbles contain a vacuum; as they implode or collapse, debris is physically removed from objects
Effective as an instrument cleaner
Efficacy: incomplete sterility

Chemical Methods
Many chemicals are available to sterilize, disinfect or sanitize
None is the “ideal” agent
Chemicals penetrate cell walls and react with cell components in various ways to destroy or inhibit growth
Many chemicals are disinfectants with varying levels of efficacy
Some are sterilants

Chemical Methods

Bacteria Viruses

Level Vegetative Acid-fast Spores Lipophilic Hydrophilic

High + + + + +

Medium + + 0 + +/-

Low + 0 0 +/- 0

Examples:

High: Aldehydes, VPHP, Chlorine-dioxide

Medium: Alcohols, Phenols, 7th generation Quats

Low: Quats

Ethylene oxide

Aldehydes

Vapor phase H2O2

Halogens

Phenols

7th generation quaternary

Alcohols

Chlorhexidine

Old generation quaternary

High-cidal

Activity

Low-cidal

Activity

Chemical Methods
Ideal chemical agent
Broad spectrum of activity
Does not stain or damage surfaces
Stable after application
Effective in a short time
Nonirritating and nontoxic to surfaces and tissues
Inexpensive and easy to store and use
Not affected by organic debris or other soil
Effective at any temperature
Nontoxic, nonpyrogenic and nonantigenic
Possesses residual and cumulative action

Chemical Methods
Soaps
Detergents
Quaternary ammonium compounds
Phenols
Aldehydes
Halogens
Chlorine and chlorine releasing compounds
Alcohols
Peroxygen compounds
Ethylene Oxide

Chemical Methods
Soaps
Anionic cleaning agent made from natural oils
Ineffective in hard water
Does not mix well with quats and decreases the effectiveness of halogens
Is not antimicrobial
Minimal disinfectant activity

Chemical Methods
Detergents
Synthetic soaps
Anionic, cationic or nonionic; anionic combined with cationic will lead to neutralization of both
Most are basic; a few are acidic
Emulsify grease and suspend particles in solution
May contain wetting agents

Chemical Methods
Quaternary Ammonium Compounds
Quats: Centrimide, benzalkonium chloride, Zephiran, Quatsyl-D, Germiphene
Effective against gm+ and gm- microorganisms and enveloped viruses
Low toxicity and generally nonirritating
Prolonged contact irritates epithelial tissues

Chemical Methods
Quaternary Ammonium Compounds
Inactivated by organic material, soap, hard water and cellulose fibers
Reduced efficacy in presence of organic debris, soap, detergents and hard water
Ineffective sporocide and fungicide
Bacteria not destroyed may clump together; those inside the clump are protected
Dissolves lipids in cell walls and cell membranes

Chemical Methods
Quaternary Ammonium Compounds
Organically substituted ammonium compounds
More effective in basic pH
Cationic detergent
Deodorizes


Chemical Methods
Phenols
Active against gm+ bacteria and enveloped viruses
Developed from phenol or carbolic acid
Synthetic phenols are prepared in soap solutions that are nontoxic and nonirritating
Prolonged contact may lead to skin lesions

Chemical Methods
Phenols
Toxic to cats because cats lack the inherent enzymes needed to detoxify the compound
May be toxic to rodents and rabbits
Not inactivated by organic matter, soap or hard water
Activity decreased by quats

Chemical Methods
Aldehydes
Active against gm+ and gm-, most acid fast bacteria, bacterial spores, most viruses and fungi
Considered to be a sterilant but may require prolonged contact

Chemical Methods
Aldehydes
Gluteraldehyde (Cidex)
Noncorrosive
Supplied as an acid, activated by adding sodium bicarbonate
Good for plastics, rubber, lenses in “cold sterilization”
Not inactivated by organic material or hard water
Irritating to respiratory tract and skin

Chemical Methods
Aldehydes
Formaldehyde (Formicide)
Aqueous solution 37% to 40% (w/v) formaldehyde
May be diluted with water or alcohol
Irritating to tissues and respiratory tract
A vapor phase surface disinfectant that slowly yields formaldehyde

Chemical Methods
Aldehydes
Biguanide (e.g. chlorhexidine gluconate [Hibitane, Precyde])
Active against gm+, most gm-, some lipophilic viruses and fungi
Efficient disinfectant, used mostly as an antiseptic
Some reduction of activity in presence of hard water and organic material
Immediate, cumulative and residual activity
Precipitates to an inactive form when mixed with a saline solution
Used as a surgical scrub and hand wash
Low toxicity

Chemical Methods
Halogens
Chlorine, iodine, fluorine and bromine
Active against gm+ and gm-, acid fast, all viruses and fungi
Iodine most common
Chlorine and chlorine releasing compounds

Chemical Methods
Halogens
Iodine
Used in solution with water or alcohol
Iodophors: iodine plus carrier molecule that acts to release iodine over time
Surgical scrub (Betadine): iodophor plus detergent
Tinctures and solutions: iodines and iodophors w/o detergent
Nonstaining and nonirritating
Inactivated by organic material
Aqueous forms are staining, irritating and corrosive to metals, especially if used undiluted

Chemical Methods
Halogens
Chlorine and chlorine releasing compounds (e.g. chlorine gas, chlorine dioxide)
Commonly available as sodium hypochlorite
Least expensive and most effective chemical disinfectant
Available chlorine equals oxidizing ability
Damages fabrics, corrosive to metals
Inactivated by organic debris
May require several minutes of contact to be effective
Skin and mucous membrane irritant if not diluted properly or rinsed well

Chemical Methods
Alcohols
Ethyl alcohol, isopropyl alcohol, methyl alcohol
Active against gm+ and gm- bacteria and enveloped viruses
Most effective when diluted to 60% to 70% (isopropyl), 705 to 80% (ethyl)

Chemical Methods
Alcohols
Used as a solvent for other disinfectants and antiseptics
Most commonly used skin antiseptic
Low cost and low toxicity

Chemical Methods
Alcohols
Irritating to tissues and painful on open wounds
Repeated use dries skin
Forms coagulum in presence of tissue fluid
Consists of a layer of tissue fluid whose proteins have been denatured by alcohol
Facilitates survival of bacteria under the coagulum
Fogs lenses, hardens plastics and dissolves some cements

Chemical Methods
Alcohols
Inactivated by organic debris
Ineffective after evaporation
Defatting agent

Chemical Methods
Peroxygen compounds (e.g. Peracetic acid)
Active against gm+ and gm-, acid-fast, fungi.
No virucidal activity
Classified as a sterilant but may not kill pinworm eggs

Chemical Methods
Peroxygen compounds (e.g. Peracetic acid)
Oxidizing agent
Reacts with cellular debris to release oxygen
Kills anaerobes
Applied as a 2% solution for 30 minutes at 80% humidity
Explosive and can damage iron, steel and rubber
Irritating to healthy tissues

Chemical Methods
Ethylene Oxide
Active against gm+ and gm-, lipophilic and hydrophilic viruses, fungi and bacterial spores
Classified as a sterilant
Effective sterilant for heat labile objects

Chemical Methods
Ethylene Oxide
EO is a colorless nearly odorless gas that diffuses and penetrates rapidly
Flammable and explosive
Toxic, carcinogenic and irritating to tissue

Chemical Methods
Ethylene Oxide
Used in a chamber with a vacuum
May be mixed with CO2, ether or freon
Used at temperatures of 21° to 60° C (70° to 140° F)
Works quicker at higher temperatures
Exposure times of 1 to 18 hours
Requires minimum relative humidity of 30% (40% is optimum
Items must be clean and dry and can be wrapped muslin, polyethylene, polypropylene or polyvinyl
Sterilized items must be ventilated in a designated area for 24 to 48 hours to dissipate residual EO

Autoclave
Advantages
Consistently achieves complete sterility
Inexpensive and easy to operate
Safe for most surgical instruments and equipment, drapes and gowns, suture materials, sponges and some plastics and rubbers
Safe for patients and personnel
Established protocols and quality control indicators are easy to access

Autoclave
Disadvantages
Staff may overestimate the ability of the autoclave
Sterility depends on saturated steam of the appropriate temperature having contact with all objects within the unit for a sufficient length of time
Requires a thorough understanding of techniques to ensure that the above occurs

Autoclave
Function
Heat is the killing agent
Steam is the vector that supplies the heat and promotes penetration of the heat
Pressure is the means to create adequately heated steam

Autoclave
Function
Complete sterilization of most items is achieved after 9 to 15 minutes exposure to 121° C (250° F)
Steam at sea level is 100° C (212° F) as pressure is increased the temperature of the steam increases
The minimum effective pressure of the autoclave is 15 pounds per square inch which provides steam at 121° C (250° F)
Many autoclaves attain 35 psi which creates steam temperature of 135° C (275° F)

Autoclave
Function
Exposure times must allow penetration and exposure of all surfaces to 121°C (250° F) steam
Exposure time is decreased by increasing pressure, which increases steam temperature

Steam Sterilization Temperature/
Pressure Chart

Temperature

Pressure(psi) °C °F Time(mins)

0 100 212 360

15 121 250 9-15

20 125 257 6.5

25 130 266 2.5

35 133 272 1

Autoclave
Types
Gravity displacement autoclave
Prevacuum autoclave

Autoclave
Types
Gravity displacement
Water is heated in a chamber
Continued heating creates pressure
Steam displaces air within the chamber forcing it out through a vent
Cycle timing begins when the temperature reaches at least 121°C

Autoclave
Types
Gravity displacement
After sufficient exposure time, steam is exhausted through a vent back into a reservoir
Air that has been sterilized within the jacket and then filtered is admitted back into the chamber to replace the exhausting steam
If the chamber is loaded improperly or there is insufficient steam, there will be air pockets remaining in the chamber that will interfere with steam penetration and result in non-sterile areas
The load must be dried within the autoclave

Autoclave
Types
Prevacuum
Usually a much larger and more costly machine
Equipped with a boiler to generate steam and a vacuum system
Air is taken out of the loaded chamber by means of the vacuum system
Steam at 121°C or more is introduced into the chamber
The steam immediately fills the chamber to eliminate the vacuum
Exposure time begins immediately
At completion of the cycle steam is vacuumed and replaced by hot, dry sterile air
Air pockets are eliminated and processing times are reduced due to the vacuum

Autoclave
Operation
Preparation of the load
Loading the chamber
Autoclave cycles

Autoclave
Operation
Preparation of the load
Linen packs
Pouch packs
Hard goods
Liquids
Contaminated objects

Autoclave
Linen packs
All instruments in packs are scrupulously cleaned and rinsed in de-ionized water
Instruments are disassembled and ratchets are left closed and unlocked
Appropriate lines are in good repair and freshly laundered
Disposable linens are not reused

Autoclave
Linen packs
A chemical sterilization indicator is included in every pack
Chemical sterilization indicators provide verification that the inside of the pack was exposed to appropriate sterilization temperatures for the appropriate length of time

Autoclave
Linen packs
The pack is wrapped using at least two layers of material
The shelf life of the sterilized pack varies with the type of the outer wrapping
Pack is sealed with autoclave tape and labeled with the date, contents and operator
Autoclave tape provides verification that the outside of the pack was exposed to appropriate sterilization temperatures

Autoclave

SHELF LIFE

Wrapper Shelf-life

Dbl wrapped two layer muslin 4 wk

Dbl wrapped two layer muslin 6 mo

heat sealed in dust covers

after sterilization

Dbl wrapped two layer muslin 2 mo

tape sealed in dust covers

after sterilization

Dbl wrapped non-woven barrier 6 mo

materials (paper)

Paper/plastic peel pouches, heat sealed 1 year

Plastic-peel pouches, heat sealed 1 year

Autoclave
Linen packs
Pack should not exceed 30 X 30 X 50 cm (12 X 12 X 20 inches) in size
Pack should not exceed 5.5 kg (12 lb) in weight
Pack should not exceed 115 kg/m3 in density

Autoclave
Pouch packs
Used for single instruments, sponges, etc.
Previous guidelines apply
Pouches are heat sealed or ends are rolled and securely taped with autoclave tape
Labeled as above

Autoclave
Hard goods
Stainless steel or other hard instruments, trays, bowls, laboratory cages and other equipment may be autoclaved without wrapping
Must be physically clean and rinsed in de-ionized water
Syringes and plungers are separated before autoclaving

Autoclave
Liquids
Contained in Pyrex flasks 3 times larger than contents require
Cover loosely with applied lid or paraffin film, or place a needle through the stopper to allow air exchange
Sterility of liquids processed in the autoclave is in question
Removing liquids from the chamber is hazardous to personnel

Autoclave
Contaminated objects
Used before disposal to decontaminate syringes, culture plates, etc., that contain biohazardous waste
Place objects in a container appropriate for disposal
Special autoclavable biohazard bags are available

Autoclave
Chamber loading
Must allow free circulation of steam
Use perforated or wire mesh shelves
Linen packs have 2.5cm to 7.5cm space between
Place multiple packs on edge instead of stacking
Paper/plastic pouches are placed in specially designed baskets that support them on edge with the paper side of each package facing the plastic side of the adjacent package
Solid bowls or basins are placed upside down or on edge
Mixed loads (hard goods and wrapped goods) have wrapped goods on upper shelf

Autoclave
Autoclave cycles
Wrapped goods
Hard goods
Liquids

Autoclave
Autoclave cycles
Wrapped goods
Has “dry” cycle that allows wrapped packs to dry
Used for most surgical packs

Autoclave
Autoclave cycles
Hard goods
Has no dry cycle
Used for trays, bowls, cages, etc. that will not be maintained in a sterile condition
Also used for flash autoclaving to quickly sterilize instruments that are needed immediately

Autoclave
Autoclave cycles
Liquids
Exhausts steam more slowly than other cycles
Used for liquids that would be forced from containers during a faster exhaust cycle

Quality Control
The effectiveness of any method of microbial control must be monitored regularly
Verification of the effectiveness of microbial control should be performed at least monthly

Quality Control
Methods
Recording thermometer
Thermocouple
Chemical indicator
Biological testing
Bowie Dick test
Surface sampling
Serology

Quality Control
Methods
Recording thermometer
Displays the temperature of the autoclave chamber
Operator observes for correct temperature during cycle
Some autoclaves are equipped with printed tape of chamber temperatures during cycle

Quality Control
Methods
Thermocouple
Used in steam and dry heat sterilization chambers
Temperature sensors are placed in the part of a test pack that is most inaccessible to steam penetration

Quality Control
Methods
Chemical indicator
Paper strips impregnated with sensitive chemicals change color when conditions of sterility are met
Used with autoclaves and ethylene oxide systems
Placed deep inside packs before sterilization

Quality Control
Methods
Biological testing
Bacterial spores are exposed to autoclave or ethylene oxide and then cultured
Recommended method for verification of proper autoclave operation in veterinary clinics

Quality Control
Methods
Bowie Dick test
Tests pre-vacuumed autoclaves for complete removal of air and uniform steam penetration
Uses a pack of uniform dimensions with a cross of autoclave tape in the center

Quality Control
Methods
Surface sampling
Surface to be tested is swabbed with a sterile applicator and transferred to a suitable media plate for growth
Surface or item is rinsed with a sterile solution, which is examined for contamination
“Contact plate” of media is touched to the surface and incubated
Recommended method for ensuring proper disinfection of surgical suites in veterinary clinics

Quality Control
Methods
Serology
The presence of viruses in the environment is monitored by serological testing of animals to determine the presence of antibodies
Animals maintained for this purpose are referred to as sentinel animals

Surgical Preparation and Instrument Care.ppt

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Fungal Presentations

Fungal Presentations from:fungalforum.com

High Dose AmBisome Treatment: what do we know?
By:V-J Anttila, Specialist in Infectious Diseases
Helsinki University Central Hospital, Finland

INVASIVE ASPERGILLOSIS
Management with liposomal amphotericin B
By:Michael Ellis

Is invasive aspergillosis hospital or community acquired: reassessing the evidence?
By:Malcolm Richardson PhD, FIBiol, FRCPath
Department of Bacteriology & Immunology
University of Helsinki, Finland

Invasive fungal infections in immunocompetent patients Does it exists ?

Antifungal combination therapy: where are we?
By:Malcolm Richardson. University of Helsinki.

Emerging fungal pathogens: clinical usefulness of new diagnostic tools

Update on glucan detection
By:Malcolm Richardson PhD, FIBiol, FRCPath
Department of Bacteriology & Immunology
University of Helsinki

Is azole prophylaxis a double-edged sword?
By:Malcolm Richardson PhD, FRCPath
Senior Lecturer in Medical Mycology
University of Helsinki, Finland

Clinical Findings in Rare and Emerging Fungal İnfections
By:Dr. Murat Akova
Hacettepe University School of Medicine
Section of Infectious Diseases
Ankara, Turkey


Liposomal amphotericin B: 20 years of clinical experience
By:Luis Ostrosky-Zeichner, MD, FACP
Assistant Professor of Medicine and Epidemiology
University of Texas Health Science Center at Houston

Antifungal and Surgical Management of a Case of Maxillary Sinus Aspergilloma
By:Riina Rautemaa
DDS, PhD, Consultant of Oral Microbiology
Helsinki University Central Hospital Maxillofacial Clinic and Laboratory Diagnostics;
and Haartman Institute, University of Helsinki, Finland


AMPHOTERICIN B NEPHROTOXICITY
By:GILBERT DERAY
PARIS , FRANCE

Changing Epidemiology:
The Importance of Broad Spectrum Therapeutics
By:Cornelia Lass-Flörl
Innsbruck Medical University

Antifungal treatment: Past and Present
By:Malcolm Richardson, PhD, FIBiol, FRCPath
University of Helsinki

Is combination antifungal therapy a viable option for the future?
By: Brian L Jones
Glasgow Royal Infirmary, UK

Fungal infections in solid organ transplantation recipients
By:Malcolm Richardson PhD, FIBiol, FRCPath
University of Helsinki and Helsinki University Central Hospital

Ten years experience of liposomal amphotericin B, AmBisome treatment in solid organ transplant recipients (SOT)

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University of Helsinki

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The body of knowledge and familiarity of use
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from University of Evansville

Bacterial Cells Shapes and Function

The Microscope

Growth and Condtions

Metabolism


Genetic Regulation

Genetic Transfer

Viruses I

Viruses II

Eukaryotic organism

More Eukaryotic organisms

Bacterial diversity

Antimicrobial Drugs

Bacterial Growth Control

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Professor and Chairman of the Department of Microbiology/Imunology
Kirksville College of Osteopathic Medicine


Prions & Viroids

The Bacteria - Structure - Function - Pathogenicity Relationships

The Viruses- General Features

The Viruses - Interference with Replication

The Bacteria - Genetics

The Bacteria - Soluble Toxins

The Viruses - Pathogenesis

The Bacteria - Metabolism & Antibiotic Sensitivity

The Viruses - Diseases

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The Fungi - Classification of Infections

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Medical Microbiology 4th Edition Edited by Samuel Baron

Medical Microbiology
Edited by Samuel Baron
Medical Microbiology 4th Edition
Edited by Samuel Baron

Section 1. Bacteriology

1. Immunology Overview
Armond S. Goldman and Bellur S. Prabhakar.

Introduction to Bacteriology

2. Structure
Milton R.J. Salton and Kwang-Shin Kim.

3. Classification
Ellen Jo. Baron.

4. Bacterial Metabolism
Peter Jurtshuk.

5. Genetics
Randall K. Holmes and Michael G. Jobling.

6. Normal Flora
Charles Patrick. Davis.

7. Bacterial Pathogenesis
Johnny W. Peterson.

8. Specific Acquired Immunity
John B. Robbins, Rachel Schneerson, and Shousun C. Szu.

9. Epidemiology
Philip S. Brachman.

10. Principles of Diagnosis
John A. Washington.

11. Antimicrobial Chemotherapy
Harold C. Neu and Thomas D. Gootz.

12. Staphylococcus
Timothy Foster.

13. Streptococcus
Maria Jevitz. Patterson.

14. Neisseria, Moraxella, Kingella and Eikenella
Stephen A. Morse.

15. Bacillus
Peter C. B. Turnbull.

16. Miscellaneous Pathogenic Bacteria
Herbert Hof.

17. Anaerobes: General Characteristics
David J. Hentges.

18. Clostridia: Sporeforming Anaerobic Bacilli
Carol L. Wells and Tracy D. Wilkins.

19. Anaerobic Cocci
Carol L. Wells and Tracy D. Wilkins.

20. Anaerobic Gram-Negative Bacilli
Sydney M. Finegold.

21. Salmonella
Ralph A. Giannella.

22. Shigella
Thomas L. Hale and Gerald T. Keusch.

23. Campylobacter and Helicobacter
Guillermo I. Perez-Perez and Martin J. Blaser.

24. Cholera, Vibrio cholerae O1 and O139, and Other Pathogenic Vibrios
Richard A. Finkelstein.

25. Escherichia Coli in Diarrheal Disease
Doyle J. Evans and Dolores G. Evans.

26. Escherichia, Klebsiella, Enterobacter, Serratia, Citrobacter, and Proteus
M. Neal. Guentzel.

27. Pseudomonas
Barbara H. Iglewski.

28. Brucella
G.G. Alton and J.R.L. Forsyth.

29. Pasteurella, Yersinia, and Francisella
Frank M. Collins.

30. Haemophilus Species
Daniel M. Musher.

31. Bordetella
Horst Finger and Carl Heinz Wirsing. von Koenig.

32. Corynebacterium Diphtheriae
John R. Murphy.

33. Mycobacteria and Nocardia
David N. McMurray.

34. Actinomyces, Propionibacterium propionicus, and Streptomyces
Geroge H.W. Bowden.

35. Leptospira
Russell C. Johnson.

36. Treponema
Justin D. Radolf.

37. Mycoplasmas
Shmuel Razin.

38. Rickettsiae
David H. Walker.

39. Chlamydia
Yechiel Becker.

40. Legionella
Washington C. Winn.

Section 2. Virology

Introduction to Virology

41. Structure and Classification of Viruses
Hans R. Gelderblom.

42. Multiplication
Bernard Roizman.

43. Viral Genetics
W. Robert. Fleischmann.

44. Effects on Cells
Thomas Albrecht, Michael Fons, Istvan Boldogh, and Alan S. Rabson.

45. Viral Pathogenesis
Samuel Baron, Michael Fons, and Thomas Albrecht.

46. Persistent Viral Infections
Istvan Boldogh, Thomas Albrecht, and David D. Porter.

47. Tumor Viruses
Joan C. M. Macnab and David Onions.

48. Epidemiology and Evolution
Frank Fenner.

49. Nonspecific Defenses
Ferdinando Dianzani and Samuel Baron.

50. Immune Defenses
Gary R. Klimpel.

51. Control of Viral Infections and Diseases
Karen L. Goldenthal, Karen Midthun, and Kathryn C. Zoon.

52. Chemotherapy of Viral Infections
Erik De Clercq.

53. Picornaviruses
Marguerite Yin-Murphy and Jeffrey W. Almond.

54. Alphaviruses (Togaviridae) and Flaviviruses (Flaviviridae)
Alan L. Schmaljohn and David McClain.

55. Togaviruses: Rubella Virus
Paul D. Parkman.

56. Bunyaviruses
Robert E. Shope.

57. Arenaviruses
Charles J. Pfau.

58. Orthomyxoviruses
Robert B. Couch.

59. Paramyxoviruses
Gisela Enders.

60. Coronaviruses
David A.J. Tyrrell and Steven H. Myint.

61. Rhabdoviruses: Rabies Virus
Charles E. Rupprecht.

62. Human Retroviruses
Miles W. Cloyd.

63. Rotaviruses, Reoviruses, Coltiviruses, and Orbiviruses
Albert Z. Kapikian and Robert E. Shope.

64. Parvoviruses
John R. Pattison and Gary Patou.

65. Norwalk Virus and Other Caliciviruses
Neil R. Blacklow.

66. Papovaviruses
Janet S. Butel.

67. Adenoviruses
Walter Doerfler.

68. Herpesviruses
Richard J. Whitley.

69. Poxviruses
Derrick Baxby.

70. Hepatitis Viruses
Arie J. Zuckerman.

71. Subacute Spongiform Unconventional Virus Encephalopathies
Clarence J. Gibbs and David M. Asher.

72. Filoviruses
Heinz Feldmann and Hans-Dieter Klenk.

Section 3. Mycology

Introduction to Mycology

General Concepts of Mycology
Michael R. McGinnis and Stephen K. Tyring.

Introduction to Mycology
Michael R. McGinnis and Stephen K. Tyring.

73. Basic Biology of Fungi
Garry T. Cole.

74. Disease of Mechanisms of Fungi
George S. Kobayashi.

75. Spectrum of Mycoses
Thomas J. Walsh and Dennis M. Dixon.

76. Antifungal Agents
Dennis M. Dixon and Thomas J. Walsh.

Section 4. Parasitology

Introduction to Parasitology

77. Protozoa: Structure, Classification, Growth, and Development
Robert G. Yaeger.

78. Protozoa: Pathogenesis and Defenses
John Richard. Seed.

79. Intestinal Protozoa: Amebas
William A. Sodeman.

80. Other Intestinal Protozoa and Trichomonas Vaginalis
Ernest A. Meyer.

81. Free-Living Amebas: Naegleria, Acanthamoeba and Balamuthia
Augusto Julio. Martinez.

82. Hemoflagellates
Rodrigo A. Zeledón.

83. Malaria
James M. Crutcher and Stephen L. Hoffman.

84. Toxoplasma Gondii
J. P. Dubey.

85. Pneumocystis Carinii
Walter T. Hughes.

86. Helminths: Structure, Classification, Growth, and Development
Gilbert A. Castro.

87. Helminths: Pathogenesis and Defenses
Derek Wakelin.

88. Schistosomes and Other Trematodes
Barbara L. Doughty.

89. Cestodes
Donald Heyneman.

90. Enteric Nematodes of Humans
John H. Cross.

91. Enteric Nematodes of Lower Animals Transmitted to Humans: Zoonoses
Doris S. Kelsey.

92. Filarial Nematodes
John H. Cross.

Section 5. Introduction to Infectious Diseases

Introduction to Infectious Diseases

93. Infections of the Respiratory System
Purushothama V. Dasaraju and Chien Liu.

94. Microbiology of the Circulatory System
Lawrence L. Pelletier.

95. Microbiology of the Gastrointestinal Tract
Sherwood L. Gorbach.

96. Microbiology of the Nervous System
Richard T. Johnson.

97. Microbiology of the Genitourinary System
Allan Ross. Ronald and Michelle J. Alfa.

98. Microbial Infections of Skin and Nails
Raza Aly.

99. Microbiology of Dental Decay and Periodontal Disease
Walter J. Loesche.

100. Bone, Joint, and Necrotizing Soft Tissue Infections
Jon T. Mader and Jason Calhoun.


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Microbiological Methods

Microbiological Methods

Making Media
Pouring Culture Plates
Sterile Technique
Inoculating Plates and Culture Tubes
Use of a Plate Counter to Estimate Microbial Population Densities

Culturing Microorganisms
Sterile Technique

* When culturing bacteria or other microorganisms, it is important to keep your work area as clean as possible.
* This prevents the introduction of other microorganisms from the environment into your culture.
* The techniques used to prevent contamination are referred to as sterile techniques.
* Start by washing your down your work or lab benches with a surface disinfectant. The most commonly used disinfectants for lab use are:
o 10% bleach (recommended by the CDC)
o 85% ethanol

Sterile Technique (2)

* Turn off any forced air heating or air conditioning units that create strong air current in your work area.
* A small room or closet that can be closed off is worth the effort to set-up if you will be doing a lot of microbial culturing.
* You can install a UV bulb in a fluorescent light fixture to surface sterilize your work bench if you have an enclosed area. Remember to leave the area when you turn on the UV light source!

Sterile Technique (3)

* All glassware should be cleaned and sterilized before you begin.
* All pipettes, spatulas, and test tube (culture) racks should also be sterilized.
* You can purchase sterile, disposable culture tubes, petri dishes, and pipettes to minimize the quantity of glassware that you have to sterilize.

Sterile Technique (4)

* Don’t forget to wash you hands after you finish cleaning and put on a pair of sterile disposable gloves before you begin.
* Once your work area is clean, your hands are clean, and your glassware is clean and sterile, don’t contaminate the work area by placing “dirty items” such as pencils, pens, notes, or books in the sterile work area.

Media Preparation
Microbiological Media

* The type of growth medium that you use is a function of the organisms that you want to culture. Use a reference book (there are many) to determine the type of medium that is best suited for your organism of interest.
* Common media include Luria Broth (LB), Nutrient Agar, Potato-Dextrose Agar (PDA), Bold’s Basal Medium (BBM)….
Luria Broth

Things to remember:
Assemble all of your chemicals in your work area before you begin.
Accurately weigh each of the dry ingredients in your culture media.
Add each dry culture medium ingredient to the culture flask.
Add distilled (or deionized) water to make the correct volume. Heat AND stir (agar will burn if it is not stirred) until all of the ingredients go into solution. When the media boils, it is ready for sterilization.
Media Sterilization

* There are two reliable methods used to sterilize microbial culture media:
o autoclave
o pressure cooker
* When using an autoclave, use the “wet” setting for sterilizing liquids (flasks, bottles, culture tubes, etc), and use the “dry” setting when sterilizing empty containers, stoppers, etc.

Media Sterilization (2)

* All liquid media should be sterilized for a minimum for 45 minutes at high temperature and pressure. Autoclaves will cycle automatically, but if you use a pressure cooker, set a timer.
* Remember not to tighten the cap or seal on any container; it will explode under high pressure and temperature!
Sterilize for 45 minutes using the wet cycle (autoclave) or at maximum pressure in a pressure cooker. Remember to cover the top of the flask or jar with aluminum foil to prevent contamination when as the media cools.
When using a pressure cooker, don’t over fill the cooker, and remember to weight your containers so they don’t fall over!
Sterilize at high temperature and pressure for 45 minutes before turning off the heat. Remember to allow enough time for the pot to heat up!

Plate Pouring Tips

* Line empty plates along the edge of the work bench.
* Open the petri dish lid at about a 30-45° angle to allow the hot liquid to cover the bottom of the dish. The thermal current created by the hot media prevents bacteria and fungal spores from landing in your clean dish.

Line your sterile petri plates along the edge of the table. Transfer hot media to a small sterile container and pour 15-20 ml of the plate media into each petri plate. The petri plate lid should be open slightly, but not completely open as this increases contamination.
* As the plates are poured, move the filled plates to the back of the table until the plates cool and congeal.
* Once the plates have cooled and the media is firm, store the plates media side-up (bottom) with the lid securely taped or the plates restacked in the manufacturer’s plastic sleeve.
* To increase the shelf-life of the plates, store in a cool, dry environment until they are used (refrigerator).


Inoculating Plates and Culture Tubes

* Clean and surface sterilize your work area as detailed in the section on Sterile Technique.
* Use either disposable inoculation loops or a metal loop that can be heat sterilized to inoculate plates, slants, and liquid culture tubes.
* If using a metal loop, be sure to cool the loop by touching the sterile cooled liquid media or the sterile culture plate before the placing the loop in your live culture. Failure to cool the loop will kill your active microbial cultures!
If gas is unavailable in your lab area, you can modify a standard Bunsen burner to use camp stove propane containers as fuel.

Inoculation of Liquid and Solid (Slant) Culture Tubes

Step 1: Remove the culture tube stopper or cap with one (do not set it down) and flame the mouth of the tube to surface sterilize the mouth. The heated tube surface will generate a thermal current that prevents contamination of the culture.

Step 2: Without setting any of the culture materials on the bench, place the sterile inoculation loop in the culture.

Step 3: Replace cap on the culture tube with the active microbes and put it in the test tube rack.

Step 4: Without setting the loop down, pick-up a sterile fresh culture tube with media with one hand, and remove the cap with the other hand.

Step 5: Flame the mouth of the clean culture tube.

Step 6: Place the inoculation loop containing the microbes in the fresh media and swirl the loop in the loop in the media to ensure even dispersal in the media.

Step 7: If using a solid media slant tube, follow steps 1-5 and then zig-zag the inoculation loop across the slanted surface of the solid media in the tube.
Step 8: Flame the mouth of the newly inoculated culture tube and replace the cap.

Step 9: Place the culture tube in test tube rack.

Step 10: Repeat until all of the sterile tubes have been inoculated. Use a fresh disposable culture loop for each tube or flame the metal loop after each tube has been inoculated.

Step 11: Incubate the culture at the recommended temperature (check with your supplier for growth requirements). If using environmental samples, incubation at room temperature will avoid the accidental culture of human pathogens.

Step 12: Dispose of all culture materials in a biohazard bag and sterilize all old cultures before pouring out cultures and washing culture tubes. Disposable culture dishes should be melted in an autoclave or pressure cooker prior to disposal.

Inoculating Petri Plates

Step 1:Remove the culture tube stopper or cap with one (do not set it down) and flame the mouth of the tube to surface sterilize the mouth. The heated tube surface will generate a thermal current that prevents contamination of the culture.
Step 2: Without setting any of the culture materials on the bench, place the sterile inoculation loop in the culture.
Step 3: Replace cap on the culture tube with the active microbes and put it in the test tube rack.
Step 4: Holding the petri dish lid at an 30-45° angle, work the inoculating loop from the outside of the plate toward the center in a zig-zag pattern that covers approximately 25% of the plate surface (think pie or pizza slice!).
Step 5: Turn the petri plate 90° to the right, dragging the inoculation loop through the last section of the plate, moving from the outside to the inside in a zig-zag motion.
Step 6: Repeat this process twice more until the entire plate surface is covered.

NOTE: If you are trying to isolate individual colonies, each turn of the dish will give you fewer microbes so that you can distinguish individual colonies.

Use of a Plate Counter for Estimating Microbial Populations

Serial Dilution of Environmental Samples or Commercial Cultures

* Serial dilution techniques should be used in the estimation of microbial population sizes.
* Serial dilution involves the use of a known amount (in ml or μl) in a known volume of liquid media.
* A one in ten dilution is made in a new liquid culture tube, and this process is usually repeated several times. The resulting cultures are dilutions of 1/10, 1/100, 1/1000, 1/10,000, for example, of the original sample.
* These cultures are plated on petri plates and incubated at the recommended temperature.

Estimating Microbial Population Size

* After the inoculated plates are incubated for the appropriate time period, the number of colonies per plate are counted.
* Population estimates are obtained by multiplying the dilution factor by the number of colonies per plate. The resulting number is a rate (function) of the initial weight or initial volume used from the environmental sample or culture (per gram soil, per ml or μl of culture).
Counting Plates

* If a commercial plate counter is not available, you can Xerox 1 mm square graph paper and use it as a grid for colony counting. You would need to estimate the total surface area (in mm2) by counting the number of squares in a dish.
* If using a commercial plate counter, touch each colony on the plate with the pen, and the cumulative number of colonies will appear on the display.

Summary

* Different media are used to culture microorganisms, be certain that you are using the appropriate media for your organism.
* Always use sterile technique to prevent contamination.
* Choose the type of media (liquid or plate) appropriate for your investigation or application.
* Sterile liquid culture tubes and media plates can be prepared in advance and stored in the refrigerator for later use (2 weeks for liquid culture tubes, 2 months for media plates).
* Liquid culture tubes, solid slant tubes, and petri plates can be used to culture microbes.
* Media and lab materials should be sterilized prior to use; an autoclave or a pressure cooker can be used in the sterilization process.
* Serial dilution and plate count techniques are used to estimate microbial populations from environmental or commercial cultures.

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Fundamental Techniques in Microbiology

Fundamental Techniques in Microbiology
Presentation lecture by:Dr Paul D. Brown

Introductory Biochemistry
Fundamental Techniques
* Microscopy
* Staining
* Aseptic technique
* Sterilization and waste disposal
* Media preparation

Microscopy
* Measurement
o Microorganisms are very small
o Use metric system
o Metre (m) : standard unit
o Micrometre = 1 x10-6 m
o Nanometre = 1 x10-9 m
o Angstrom = 1 x10-10 m

Terms Relevant to Microscopy
* Total Magnification
o Eyepiece x objective lens
* Resolution
o Ability of the lens to distinguish two points as separate
o Optimal RP achieved with blue light
o Theoretical limit for light microscope is 0.2 m
* Refractive Index (η)
o Measurement of relative velocity at which light passes through a material.
o η= 1.0 in air
o η (Oil) = η (glass) = up to 1.5
Resolving Power
Transmission electron microscope
Scanning electron microscope
Light microscope
Human eye
R.P. in Angstroms
Resolving Power
Optical Instrument

Types of Microscopes
* Simple: one lens
* Compound: more than one lens

The Compound Microscope
* enters the eye
o sees virtual, inverted image
* further magnif. by ocular
* forms magnified real image
* enters objective
* focuses light on object
* light enters condenser

Objectives

* 10X Scanning Find the object
* 40X High-Dry Focus the object
* 100X Oil immersion Fine focus

The Condenser
* Functions
o Focus light on object plane
o Ensure adequate intensity
* Height of condenser controls
o Uniformity of brightness
o Contrast (minimises “stray light”)
o (Indirectly) angle of light entering objective

Use of Immersion Oil
Condenser Iris Diaphragm
Bright-field Microscope
* Contains two lens systems for magnifying specimens
* Specimens illuminated directly from above or below
* Advantages: convenient, relatively inexpensive, available
* Disadvantages: R.P 0.2 m at best; can recognize cells but not fine details
* Needs contrast. Easiest way to view cells is to fix and stain.

Different magnifications
Special Microscopy Applications
* Dark Field
* Phase Contrast
* Fluorescence
* Electron Microscope
* special condenser diaphragm
o occludes direct light, passes wide angle light
o angle too wide to enter objective
Phase Contrast Microscopy
Fluorescence Microscopy
Electron Microscopy
Stains and Staining
Simple Stains
Differential Stains
* Gram stain
o Crystal violet: primary stain
o Iodine: mordant
o Alcohol or acetone-alcohol: decolourizer
o Safranin: counterstain
o Gram positive: purple
o Gram negative: pink-red

Staphylococcus aureus
Escherichia coli
Gram stain – distinguishes Gram+ from Gram -
* Acid-fast stain
Special Stains
* Capsule stain
* Flagella stain
* Spore stain (Schaeffer-Fulton)

Aseptic Technique
* First requirement for study of microbes
o pure cultures, free of other microbes
* Maintain a clean environment; work close to the flame

Streak plate method of isolation
Sterilization and Waste disposal
Culture media formulation
Types of media

* General purpose
o Allows growth of most bacteria, e.g., Nutrient agar
o Includes organic C, N, vitamins
o May have undefined components e.g., yeast extract, peptone
* Defined
o All components are pure compounds, not mixtures such as yeast extract
o E.g., glucose + (NH4)2SO4 + minerals for E. coli

* Selective
o Favours one organism and limits growth of others
o Lacks some factor(s)
+ E.g., fixed N, to select for N2-fixing bacteria
o Selective toxicity
+ E.g., bile salts to select for Enterobacteriaceae
* Selective via incubation conditions
o E.g., gas composition (e.g., N2, 5% CO2, O2), temperature
* Differential
o Different bacteria/groups give different responses
o E.g., MacConkey agar: has lactose + peptone + indicator (neutral red)
+ lactose fermenters - acid - pink colour
+ non-lactose fermenters use peptone - neutral or alkaline - colourless

Enrichment Techniques

* Increase proportion of desired physiological class
o E.g., N2-fixers; cellulose-decomposers; photosynthetic bacteria
* Culture mixed population in selective medium and/or conditions
o E.g., fixed N-free; cellulose as sole carbon, energy source; anaerobic conditions in light, without organic C
* Sample treatment
o E.g., boil to kill vegetative cells, leaving spores

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