Microbiology specimen collection

and Normal Skin Flora

John Santangelo

The different layers of the skin are important defenses of the skin.

Cross-section of skin

Flora refers to bacteria

Infection and Disease A.Definitions

B. The Normal Flora of Humans C. Generalized Stages of Infection D. Virulence Factors and Toxins

Definitions
Disease and Infectious Disease Disease Any deviation from a condition of good health and well-being.

Infectious Disease A disease condition caused by the presence or growth of infectious microorganisms or parasites.

Definitions
Pathogenicity and Virulence

Pathogenicity The ability of a microbe to cause disease. This term is often used to describe or compare species.
Virulence The degree of pathogenicity in a microorganism. This term is often used to describe or compare strains within a species.

Definitions
Acute infection vs. chronic infection

Acute Infection An infection characterized by sudden onset, rapid progression, and often with severe symptoms.
Chronic Infection An infection characterized by delayed onset and slow progression.

Definitions
Primary infection vs. secondary infection Primary Infection An infection that develops in an otherwise healthy individual. Secondary Infection An infection that develops in an individual who is already infected with a different pathogen.

Definitions
Localized infection vs. systemic infection Localized Infection An infection that is restricted to a specific location or region within the body of the host. Systemic Infection An infection that has spread to several regions or areas in the body of the host.

Definitions
Clinical infection vs. subclinical infection
Clinical Infection An infection with obvious observable or detectable symptoms.

Subclinical Infection An infection with few or no obvious symptoms.

Definitions
Opportunistic infection

An infection caused by microorganisms that are commonly found in the hosts environment.
This term is often used to refer to infections caused by organisms in the normal flora.
Waiting for the opportunity to cause infection

Definitions
The suffix -emia
Meaning presence of an infectious agent Bacteremia = Presence of infectious bacteria. Viremia = Presence of infectious virus.

Fungemia = Presence of infectious fungus.

Septicemia = Presence of an infectious agent in the bloodstream. (Blood Poisoning)

Definitions
The suffix -itis Meaning inflammation of Pharyngitis = Inflammation of the pharynx. Endocarditis = Inflammation of the heart chambers. Gastroenteritis = Inflammation of the gastointestinal tract. (GIT)

Definitions

Epidemiology The study of the transmission of disease. Communicable Disease A disease that can be transmitted from one individual to another. Noncommunicable Disease A disease that is not transmitted from one individual to another. Contagious Disease A communicable disease that is easily spread from one individual to another.

The study of the geographic and socialeconomic distribution of disease.

Definitions
Endemic Disease A disease condition that is normally found in a certain percentage of a population. Epidemic Disease A disease condition present in a greater than usual percentage of a specific population. Pandemic Disease An epidemic affecting a large geographical area; often on a global scale.

Definitions
Reservoir of Infection The source of an infectious agent.

Carrier An individual who carries an infectious agent without manifesting symptoms, yet who can transmit the agent to another individual.
Fomites Any inanimate object capable of being an intermediate in the indirect transmission of an infectious agent.

Definitions
Animal Vectors
An animal (nonhuman) that can transmit an infectious agent to humans. Two types: mechanical and biological Biological animal vectors: The infectious agent must incubate in the animal host as part of the agents developmental cycle; eg, the transmission of malaria by infected mosquitoes.

Mechanical animal vectors: The infectious agent is physically transmitted by the animal vector, but the agent does not incubate or grow in the animal; eg, the transmission of bacteria sticking to the feet of flies.

Definitions Direct Mechanisms of Disease

Transmission Directly From Person to Person.

Examples: Direct Skin Contact Airborne (Aerosols)

Definitions Indirect Mechanisms of Disease Transmission Examples: Food & Waterborne Transmission. Fomites. Animal Vectors.

The Normal Flora of Humans

Types of Symbiosis
Mutualism A symbiotic relationship in which both species benefit.

Commensalism A symbiotic relationship in which one species benefits, and the other species is neither helped nor harmed.

The Normal Flora of Humans Normal flora is present in:

- skin

- upper respiratory tract - oral cavity - intestine, especially large intestine - vaginal tract - Very little normal flora in eyes & stomach

The Normal Flora of Humans

Notably absent in most all internal organs

Absent in: - lower respiratory tract - muscle tissue - blood & tissue fluid - cerebrospinal fluid - peritoneum - pericardium - meninges

The Normal Flora of Humans

Benefits of the normal flora Nutrient production/processing. e.g. Vitamin K production by E. coli. Competition with pathogenic microbes.

Normal development of the immune system.

Normal flora and opportunistic infections.

Generalized Stages of Infection 1. Entry of Pathogen - Portal of Entry 2. Colonization - Usually at the site of entry 3. Incubation Period - Asymptomatic period Between the initial contact with the microbe and the appearance of the first symptoms.

Generalized Stages of Infection

4. Prodromal Symptoms - Initial Symptoms 5. Invasive period - Increasing Severity of Symptoms Fever Inflammation and Swelling Tissue Damage Infection May Spread to Other Sites Acme (Fastigium)

Generalized Stages of Infection

6. Decline of Infection

5. Convalescence

Virulence Factors and Toxins

State of the Host Immune System. Number of Pathogenic Cells encountered by the Host. Infectious Dose.

Virulence Factors and Toxins

Enzymatic Virulence Factors Examples: Coagluase (Staphylococcus aureus) Streptokinase (Streptococcus pyogenes) Hyaluronidase (Many pathogens) Collagenase (Many pathogens) Leucocidin (Many pathogens) Hemolysin (Many pathogens)

Virulence Factors and Toxins

Adhesion Factors Examples: Protein A (Staphylococcus aureus) Protein M (Streptococcus pyogenes)

Virulence Factors and Toxins

Exotoxins
A type of bacterial toxin with the following properties: - May be produced by either gram-positive or gramnegative bacteria. - Is secreted by the bacteria. - The action of the exotoxin does not necessarily require the presence of the bacteria in the host. - Most exotoxins are peptide or protein. - Most exotoxins are heat sensitive (exception: enterotoxin of Staphylococcus aureus)

Staphylococcus Aureus is also known as Golden Staph

Virulence Factors and Toxins

Exotoxins (cont.) Classes of exotoxins: Neurotoxic, cytotoxic, or enterotoxic exotoxins Neurotoxins: Interfere with proper synaptic transmissions in neurons. Cytotoxins: Inhibit specific cellular activities, such as protein synthesis. Enterotoxins: Interfere with water reabsorption in the large intestine; irritate the lining of the gastrointestinal tract.

Virulence Factors and Toxins

Endotoxins A type of bacterial toxin having the following properties: Produced only by gram-negative bacteria

Endotoxins are a component of the gramnegative cell wall The action of endotoxin requires the presence of the bacteria in the host. The endotoxin may be released from the cell wall as the cells die and disintegrate

Virulence Factors and Toxins

Endotoxins (cont.) Endotoxin is composed of Lipid A: Part of the lipopolysaccharide layer. Mode of action: Irritation/inflammation of epithelium, GI irritation, capillary/blood vessel inflammation, hemorrhaging.

Normal Skin Flora

The normal skin flora is made up of the bacteria that normally live on the skin when the skin is healthy.

These bacteria, which are harmless, actually prevent other harmful bacteria and fungus from growing on the skin.
These normal bacteria are disturbed with over washing with antibacterial soaps and the use of antibiotics. The purpose of antibacterial soap is to kill bacteria on our skin that can cause bacterial infections.

Unfortunately it also kills the Normal Flora that prevents potential harmful bacteria causing infection.

Our normal flora acts as a protective barrier

The normal flora influences the anatomy, physiology, susceptibility to pathogens, and morbidity of the host.
The composition of the dermal micro flora varies from site to site according to the character of the microenvironment. A different bacterial flora characterizes each of three regions of skin: (1)axilla, perineum, and toe webs; (2) hand, face and trunk; (3) upper arms and legs.
Morbidity the degree or severity of a disease,

Skin sites with partial occlusion (axilla, perineum, and toe webs) harbour more micro-organisms than do less occluded areas (legs, arms, and trunk).

Susceptibility increases where there is a break in the skin. (scratches, mat burns, skin blemishes)

Poor hygiene also increases the hosts susceptibility.

Continued exposure to foreign skin flora promotes disease.

Identification of Bacteria 1. The site of origin. 2. The type of agar plate it grows on. The temperature. 3. The shape and smell of the colony.

4. Whether it haemolyses a blood plate or not.

5. The type of plate used. 6. Chemical tests. (fementers etc) 7. Aerobe or anaerobe. 8. Gram Stain. 9. Microscopy. 10. Special stains. (ZN for TB) etc.

Swabs
John Santangelo

This nutrient contains charcoal and therefore is black. In most swabs, the cotton tip contains the charcoal and not the nutrient.

It is used when swabbing for Gonorrhoea.

The charcoal absorbs the oleic acid in the cotton wool which would otherwise kill the gonorrhoea.
Nutrient agar with charcoal

Swab Stick

Swab for general bacterial infections. It contains nutrient agar only, which keeps the bacteria alive and prevents multiplication.

The swab before opening

After opening
Wire, plastic & wood shafts are used.

Swabs for Bacterial (red)

(sometimes yellow)

Viral Cultures (green)

After replacing the viral swab into the container, squash the bottom end with your fingers to break the glass to release the fluid. (Viral swab only)

Glass containing fluid & nutrient

Flexible wire swab used for Nasopharyngeal and Urethral Swabs

Nasopharyngeal swab for Whooping cough. (Pertussis)

Urethral swab for gonorrhoea & Chlamydia.

4N6 DNA SWAB - FORENSIC COLLECTION SYSTEM

Convenient kit format for collection of DNA evidence at crime scenes or from suspects and victims. Can be used for collection of buccal swab samples and blood stains.
Just peel open the pouch, collect the sample and snap the applicator shaft into the cuvette-tube provided. Snaps here

Plastic container & lid

When swabbing a blister caused by Herpes, the blister must be broken and the base of the opened blister swabbed applying pressure in a circular motion. If this technique is not used, viral recovery will not be achieved. Herpes is a Virus so use a Green Swab.

Herpes Blisters

Herpes Zoster (Shingles) Chicken Pox Virus

MSU Mid Stream Urine also called UMCS Urine Micro Culture and Sensitivity Why do an MSU? For a Urinary Tract Infection (UTI) What is a UTI? An infection of the Urinary Tract.

i.e. kidney, ureters, bladder & urethra

It usually only involves the bladder but any or all of the above can be involved.

Causes of a UTI
The most common cause of UTI is bacteria from the bowel that live on the skin near the rectum which can spread and enter the urinary tract through the urethra. Once the bacteria enter the urethra they travel upwards causing infection in the bladder and sometimes other parts of the urinary tract.
The most common bacteria to cause a UTI is the normal bacteria that live in the bowel, i.e. E Coli Females are more often effected because of a short urethra.

Having a bath rather than a shower makes it more likely to get a UTI.
The bacteria enter by capillary action.

Performing a Mid Stream Urine

The patient is given a Yellow Top container
The container should be sterile but most pathology companies do not use sterile containers
The patient is also given a sterile water swab to wipe the area from Front to Back. A small amount of urine is passed into the toilet to clear the urethra of any bacteria that would contaminate the specimen. Then, the second catch is collected into the yellow top container. Instruct the patient not to touch the inside of the container or lid. It is best not to do an MSU during a menstrual cycle.

NOTE: The Urine can be collected at home or in the collecting room. If collected at home, instruct the patient to store it in a cool place and bring it in as soon as possible. Leaving a urine in a warm place for long periods will cause the bacteria, if present, to multiply and give a false indication that a more severe infection is present. Leaving the urine in a cool place will slow down the multiplication of the bacteria. It should, ideally be brought into the collection room within an hour of collection.

Dipsticks
Used for detecting: Protein Blood Nitrites (infection)

Ketones
Glucose pH

Diabetics

White Cells (Leukocytes)

Bilirubin Haemoglobin

Chlamydia Infection
The second morning urine is collected for Chlamydia. A urethral swab is also done but the second morning urine is taking over.
Flexible wire swab for the urethra

Sputum
The mucus and other matter brought up from the lungs, bronchi and trachea that one may cough up, spit or swallow. Sputum is borrowed directly from the Latin to spit. It is also called expectoration. Make sure it is not saliva. An early morning specimen is recommended as it yields the best specimen. Because of lack of activity while sleeping, more secretions accumulate in the lungs.

The sputum must be taken to the collection centre within 2 hours.

A sputum is done to detect infection in the lungs or bronchial tubes.

The following instructions are given to the patient when TB (Tuberculosis) is requested.
TB is also called Acid Fast Bacilli (AFB)

Three early morning sputum specimens are collected and taken to the collection centre each day.
The container need not be sterile as bacteria other than AFB is destroyed with Sodium Hydroxide. The same instructions are used when sputum cytology is requested. Sputum Cytology is looking for abnormal cells, usually cancer cells.

Faeces
Faeces are collected for the following reasons:

Bacteria
Parasites, known as Ova, Cysts & parasites (OCP) especially when diarrhoea is present.

Blood (usually Occult blood) also known as Faecal Occult Blood (FOB)
Fat (for malnutrition)

Mucus
Fungi & Yeasts

Cerebrospinal Fluid (CSF)

The cerebrospinal fluid surrounds the Brain and Spinal Cord.

The CSF is tested for the following:

Bacteria or viruses and Encapsulated yeasts when meningitis is suspected.

Protein when a tumour is suspected.

Glucose when a bacterial infection is suspected. It is important to do a blood glucose as well. Two tubes are taken labelled 1 and 2 Number 2 is used as it is not contaminated with blood from the first traumatic spinal tap.

After a cerebral Haemorrhage, (bleeding into the brain), red cells will be found in the CSF. If the Red Cells have haemolysed (broken up), the CSF will have a pinkish to red tinge according to the degree of haemolysis.

This is called, Xantchromia.

Other Specimens
Joint fluid for Uric Acid Crystals as occurs in Gout.

Joint fluid for TB.

Pleural fluid Peritoneal fluid

Skin Scrapings
Nail Clippings Eye, Ear and Nasal Swabs Buccal scrapings for DNA

Pathology Report

Dogs are not allowed on the beach

The End

John Santangelo E Mail: saint547@optusnet.com.au