Unknown Culture Report

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Objective

The objective of this lab was to perform various tests using knowledge learned in class, to decipher an unknown culture by utilizing different agar plates and test tubes to select, differentiate and bio chemically test the unknown. This experiment was performed in two consecutive periods.

Result

The unknown culture #31 was found to be Serratia marcescens. Its striking feature was its production of red pigment observable on H2S test tubes.

A schema diagram of how I identified the unknown is at the end of this report.

Period 1

In period 1 the following materials were obtained and procedures were performed.

Equipment/Materials

Unknown test tube culture #31 was selected.

In addition to Bunsen burner and inoculating loop, different medias to culture and grow the unknown were assigned. This includes.

  • TSA agar plate

  • McConkey plate

  • Mannitol Salt Agar

  • Starch Plate

  • SIM tube

  • Citrate tube

  • Urea tube

  • Lactose

  • Sucrose

  • Glucose

  • Voges-Proskauer

Procedures

The unknown culture was then inoculated on the agar plates and test tubes to culture and grow the unknown microorganism using best standard practices. For example:

In the case of Mannitol Salt Agar the streak plate technique was used by performing the following procedures.

  • An inoculating loop is sterilized by flaming it using a Bunsen burner.

  • The inoculating loop is cooled down.

  • The cap of the unknown culture was removed from the top of the tube and was also flamed for sterilization.

  • The inoculating loop was inserted in the unknown tube to pick up culture.

  • The the top part of the tube was then passed again over the flame.

  • The culture is streak on the plate using the streak plate technique and aseptic technique.

Similar method was used on the test tubes without using the streak plate method. That is,

culture was picked up using the aseptic technique and introduced on each of the test tubes.

After these procedures, the culture was disposed and the inoculated plates and test tubes were placed in the refrigerator until the next period.

Second Period and Results

Additional Materials

  • Slides

  • Microscope

  • Gram Stain Reagents(Crystal violet, Gram’s iodine, Ethyl alchol, Sufranin, Distilled water)

The inoculated plates and tubes were analyzed. From the Mannitol Salt Agar (MSA) test, my unknown was discovered to be gram negative since it was not a lactose fermenter.

After gram staining, my unknown was observed and reaffirmed that it was gram negative since it stained red which was the color of the counterstained sufranin. Furthermore, it was obvious from the microscope at oil immersion power that it had short rods (bacilli) characteristics.

Once gram negative and bacilli shape were known, possible microorganisms could be identified based on class handout including.

E coli

Enterobacter aeruginosa

Serratia Marcescens

Proteus Vulgaris

Proteus Mirabilis

Pseudomonas Aeruginosa.

Also from the MSA plates the unknown culture was observed to have the following colony description.

Whole Colony Appearance

Circular

Margin Edge

Entire

Elevation

Convex

Below is the result of bio chemical tests. Some tests were unnecessary to deduce the unknown microorganisms. A flow chart is included detailing my thought processes and how I arrived at the unknown which was Serratia marcescens.

Mannitol Salt Agar

Negative

Lactose

Negative

Sucrose

Positive

Glucose

Positive

H2S

Negative (red pigment)

Indole

Negative

Motility

Positive

Urea

Negative

Voges-Proskauer

Positive

Starch

Negative

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Chapter 7 The Control of Microbial Growth

Chapter 7 The Control of Microbial Growth

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The Terminology of Microbial Control

  • Aseptic surgery is surgery performed under sterilized condition to prevent microbial contaminantion to the patient.

  • Sterilization is the removal of all forms of microbial organisms.

  • Commercial sterilization is a limited heat treatment, just enough to destroy microbes, and not to degrade the quality of the foods.

  • Disinfection is the destruction of vegetative pathogens on an inert surface or substance by using chemicals.

  • Antisepsis refers to the destruction of microbes in living tissue.

  • Degerming refers to the removal of not killing of microbes in a limited area.

  • Sanitization is the lowering of microbial counts on eating utensils.

  • Biocide or germicide refers to the killing of microbes.

  • Bacteriostasis refers to the inhibition of bacteria and not killing them.

  • Sepsis refers to microbial contamination.

  • Asepsis refers to the absence of significant microbial contamination.

The Rate of Microbial Death

Influential Factors of Antimicrobial Treatments

  1. The number of microbes.

  • The more microbes the longer it take to eliminate them.

  1. Environmental Influences.

  • Presence of organic matter (blood, vomit, feces) inhibits antimicrobial actions.

  • Nature of suspending medium (high fats and proteins) protects microbes.

  • Heat is more effective under acidic conditions.

  1. Time of exposure.

  • More-resistant microbes require longer antimicrobial exposure.

  1. Microbial characteristics

  • Microbial characteristics affect chemical and physical control methods.

Actions of Microbial Control Agents

Alteration of Membrane Permeability

  • Microbial Control Agents target plasma membrane damaging its lipids causing it to leak.

Damage to Proteins and Nucleic Acids

  • Antimicrobial agents denatures microbial proteins and damages DNA and RNA.

Physical Methods of Microbial Control

  1. Heat

Moist Heat (kills microorganisms by denaturation)

  • Boiling

  • Autoclave (steam under pressure; preferred method; complete sterilization; 121 deg C for 15 min)

  • Pasteurization (incomplete sterilization; classical method: 63 deg C for 30 min; high temp method: 72 deg C for 15 sec; ultra high temp: 140 deg C for 1 sec; thermoduric organism survives.

Dry Heat (Kills by oxidation)

  • Flaming (Such as the one used in lab)

  • Hot-air sterilization (Items to be sterilized are placed in the oven at 170 deg C for 2 hours.

  1. Filtration

  • Removes microbes

  • HEPA (high-efficiency particular air) removes microorganisms larger than .4 um.

  • Membrane filters have become popular in industrial and laboratory use.

  1. Low Temperatures

  • Psychrotrophs grow at refrigerator temperature.

  1. High Pressures

  • Used usually to preserved fruit juices. Endospores are resistant to high pressures.

  1. Desication

  • In the absence of water, microorganisms cannot grow or reproduce but can remain viable for years.

  1. Osmotic Pressure

  • Cause osmolysis

  • Molds and yeasts are more capable than bacteria of growing in materials with low moisture or high osmotic pressure.

  1. Radiation

  • Ionizing radiation includes gamma rays, X rays, or high energy electron beams.

  • Nonionizing radiation include UV (ultraviolet) light.

Chemical Methods of Microbial Control

Types of Disinfectants

  1. Phenol and Phenolics

  • Phenols rarely used as antiseptic or disinfectant because its skin irritation and disagreeable odor factors.

  • Phenolics is a derivative of phenols altered to have less irritation and disagreeable odor factors.

  • Cresols is one of the most frequently used phenolics.

  1. Bisphenols

  • Bisphonol is a derivative of phenols containing two phenolic groups connected by a bridge

  • Hexachlorophene is one bisphenol used in hospital surgical and microbial control procedures. Also used in prescription lotion, pHisoHex.

  • Triclosan is another bisphenol use in bacterial soaps and some toothpastes.

  1. Biguanides

  • Chlorhexidine is a member of biguanides use for microbial control on skin and mucous membranes.

  • With detergent or alcohol it is used for surgical hand scrubs and preoperative skin preparation in patients.

  1. Halogens

  • Composed of iodine and chlorine

  • Iodine is available as tincture (solutions in acqueous alcohol) and iodophor (combination of iodine and organic molecule such as betadine)

  • Chlorine as gas (Cl2) is germicidal disinfectant because of its production of hypochlorous acid when it is added to water.

  • Sodium hypochlorite is used as household disinfectant such as (Clorox).

  • Calcium hypochlorite is used to disinfect dairy equipments and restaurant utensils.

  • Chlorine dioxide is used for area disinfection to kill endospores of anthrax bacterium.

  • Chloramines consist of chlorine and ammonia used as disinfectants, antiseptics and sanitizing agens.

  1. Alcohols

  • Alcohols such as ethanol and isopropanol kill bacteria and fungi but not endospores and non-enveloped virus.

  1. Heavy Metals and their Compounds

  • Heavy metals includes silver, mercury and copper.

  • Silver nitrate used to be apply to the eyes of the newborn to prevent gonococcal infection. Now, antibiotics are used.

  • Silver-sulfadiazine is available as topical cream for use on burns surfaces.

  • Mercuric chloride is used as bacteriostatic but discontinued because of its toxicity.

  • Copper sulfate destroys green algae in pools, tank and water supply. Also used in paints to prevent the formation of mildew.

  • Zinc chloride is present in mouth washes and used as anti-fungal in paints.

  1. Surface-Active Agents

  • Surface-active agents includes soaps and detergents to decrease surface tension of molecules of liquids.

  • Soaps and detergents are good degerming agents that removes microbes by emulsification.

  • Acid-anionic sanitizers are important in the cleaning of dairy utensils and equipments because of the negative portion (anion) of the molecule.

  • Quaternary ammonium compounds are widely used because of its bactericidal effects on gram-positive bacteria and it positively (cation) charged molecules.

  1. Chemical Food Preservatives

  • Sorbic acid and sodium benzoate prevent molds from growing in certain acidic foods such as cheese and soft drinks.

  • Sodium nitrate and sodium nitrite are added to many meat products such as ham, bacon, hot dogs to preserve the red color and prevent botulism endospore formation such as Clostridium botulinum.

  1. Antibiotics (Food usage)

    • Nisin is added to cheese to inhibit the growth of certain endospore forming spoilage.

    • Natamycin is antifungal antibiotics used in cheese.

  1. Aldehydes

  • Among the most effective antimicrobials

  • Formaldehyde gas is used to preserve biological specimen and floor disinfectant with irritative odor.

  • Glutaraldehyde is less irritating, more effective and used to disinfect hospital instruments including respiratory theraphy equipment.

  1. Gaseous Chemosterilizers

  • Ethylene oxide kills all microbes and endospores but requires longer exposures period 4 to 18 hours and used to sterilize medical supplies and equipments.

  1. Peroxygens (Oxidizing Agents)

  • Hydrogen peroxide is an antiseptic found in household cabinets and in hospital supply rooms. Also used in hair coloring products.

Microbial Characteristics and Microbial Control

Decreasing Order of Resistance to Chemical Biocides

  1. Prions

  1. Endospores of bacteria

  • Bacillus, Clostridium

  1. Mycobacteria

  • Mycobacterium

  1. Cysts of protozoa

  1. Vegetative protozoa

  1. Gram-negative bacteria

  1. Fungi, including most fungal spores

  1. Viruses without envelopes

  1. Gram-positive bacteria

  1. Viruses with lipid envelopes.

Mannitol Salt Agar (MSA) Test

Mannitol Salt Agar (MSA) Test

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Procedure

  • Innoculate culture on the MSA plate using the streak method.

Important Properties

  • Composed of NaCl, mannitol and phenol red as pH indicator

  • Both selective and differential

  • Selective since it allows only the growth of high salt or saline loving organisms.

  • Differential since manitol fermenter organisms change the red pH indicator color (neutral) to yellow (acidic).

  • Non-mannitol fermenters do not change the color of the media.

  • Usually allows the growth of Staphylococci especially for differentiating Staphylococcus aureus from others.

Expected Results

  • Mannitol fermenters includes: Staphylococcus aureus

  • Non-mannitol fermenters includes: Staphylococcus epidermidis

  • Positive growth but non-mannitol fermenters includes: Micrococcus luteus

  • Negative growth includes: Escherichia coli, Pseudomonas aeruginosa

Example

MacConkey Agar Test

MAC (MacConkey) Agar Test

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Procedure

  • Innoculate culture on the MAC plate using the streak method.

Important Properties

  • Composed of bile salts and lactose

  • Both selective and differential

  • Contains bile salts and crystal violet as its selective agents inhibiting gram positive bacterial growth.

  • Differential since lactose fermenter organisms (coliforms) give red color.

  • Non-lactose fermenters give colorless colonies.

Expected Results

  • Lactose fermenters includes: Escherichia coli, Enterobacter aerogens

  • Non-lactose fermenters includes: Salmonella typhimurium(non-coliform), Staphylococcus aureus (non-enteric), Proteus mirabilis

Example

Chapter 16 Innate Immunity: Nonspecific Defense of the Host

Chapter 16 Innate Immunity:

Nonspecific Defenses of the Host

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Immunity or resistance is the body’s ability to ward off disease from microbes and protect our selves from environmental agents(pollens, drugs, foods and chemicals).

Susceptibility is the body’s vulnerability to diseases and lack of immunity.

Overview of the Body’s Defenses

First Line of Defense (Innate/Nonspecific Immunity)

  • Skin

  • Mucus membrane

  • Microbiota

Second Line of Defense (Innate/Nonspecific Immunity)

  • Natural Killer Cells

  • White Blood Cells (Phagocytic)

  • Inflammation

  • Fever

  • Antimicrobial substances

    1. complement

    2. interferon

Third Line of Defense (Adaptive/Acquired Immunity)

  • T cells

  • B cells

  • Antibodies

The Concept of Immunity

Innate (nonspecific) immunity refers to the defenses present at birth such as skin and mucus membrane

Adaptive (specific) immunity refers to the defenses that have specific microbial recognition that acts against the microbes when the first line of defense is breached, such as T and B cells and antibodies.

First Line of Defense

Physical Factors

1. Skin (Intact)

  • Epidermis is the outer, thinner portion directly in contact with the external environment.

    • Top layer sheds periodically to remove microbes

    • Contains keratin as protective protein.

  • Dermis is the inner and thicker portion consisting of connective tissues.

  • Keratin is the protective protein of the epidermis layer.

2. Mucous Membranes and other physical factors

  • Consist of epithelial and underlying connective tissue layers such as respiratory and gastrointestinal tracts.

  • Less protection than skin but nonetheless inhibit microbial entrance

  • Mucus is a thick viscous fluid secreted by mucous membrane to lubricate tracts.

  • Treponema pallidum survives in mucus and can penetrate the membranes.

  • Lacrimal apparatus (lacrimal glands, tears, canals, ducts) wash away keep microorganisms from settling on the eyes.

  • Saliva produce by salivary glands dilutes microorganism, wash them away from teeths and mucous membranes of the mouth

  • Nose hairs filters inhaled microorganisms

  • Cillia in the lower respiratory tract help propels out toward the throat (ciliary escalator) microorganisms that have become trapped.

  • Epiglottis that covers the larynx (voice box) during swallowing prevents microorganisms from entering the lower respiratory tract.

  • Urine cleanse urethra

  • Vaginal secretions move microorganisms out.

  • Defecation and vomiting expel microbes

3. Normal Microbiota

  • Normal Microbiota are opportunistic microorganisms that inhabit the body and helps ward off and do not cause disease under normal conditions.

  • Normal microbiota in the vagina secrete acidic substance to prevent Candida albicans from causing vaginitis

  • Escherichia coli secretes bacteriocins that inhibit the growth of Salmonella and Shigella.

Chemical Factors

1. Sebum

  • Oily substance produce by sebaceous glands of the skin to form protective film on the skin surface and to prevent hair from drying and becoming brittle.

2. Low pH (3 and 5)

  • Caused by secretions of lactic acid and fatty acid prevents growth of many microorganisms.

3. Lysozyme

  • Contains in the perspiration breaks down cells walls of gram-positive bacteria and to lesser extent gram-negative bacteria.

4. Gastric Juice (pH 1.2-3.0)

  • Gastric juice which is a mixture of hydrochloric acid, enzymes and mucus produced by stomach glands destroy bacteria and their toxins.

  • Exception is Clostridium botulinum and Staphylococcus aureus.

  • Helicobacter pylori neutralizes stomach acid and grow to cause ulcers and gastritis.

Second Line of Defense

Leukocytes (White Blood Cells)

Granulocytes

  • Granulocytes have large granules in their cytoplasm that can be seen under a light microscope

1. Neutrophils

  • Stains a pale lilac with a mixture of acidic and basic dyes and consist of 2 to 5 lobes.

  • Phagocytic, motile, active in the initial stages of infection.

  • Have the ability to leave the blood, enter an infected tissue and destroy microbes and foreign particles.

2. Basophils

  • Stains blue-purple with the basic dye methylene blue

  • Phagocytic

  • Release substances such as histamine that is important in inflammation and allergic response.

3. Eosinophils

  • Stain orange or red with the basic dye eosin.

  • Phagocytic and have the ability to leave the blood.

  • Produce toxic proteins against certain parasites such as helminthes.

Agranulocytes

  • Have granules but are not visible under a light microscope.

1. Monocytes

  • Are not phagocytic until they leave the blood and mature into macrophage and become the best phagocyte in the tissue.

2. Lymphocytes

  • 3rd line of defense

  • Play a key role in the adaptive immunity.

  • Natural killer cells

  • T cells

  • B cells

Phagocytes

  • Phagocytosis is the ingestion of microorganisms and other particles by a cell.

  • Phagocytes are collective cells that perform phagocytosis.

  • Phagocytes are activated by cytokines, lipid A and lipopolysaccharides (LPS).

The Mechanisms of Phagocytosis

1. Chemotaxis and adherence of microbe to phagocyte

  • Microbe surface attaches to the plasma membrane of the phagocyte.

2. Ingestion of microbe by phagocyte

  • Pseudopods engulf the microbe

3. Formation of a phagosome

  • Phagosome surrounds the microbe.

4. Formation of phagolysosome

  • Fusion of phagososome and lysosome.

5. Digestion of ingested microbe by enzymes.

6. Formation of residual body containing indigestible material.

7. Discharge of waste materials.

Inflammation

  • Defensive response triggered by damage to the body’s tissue.

  • It is characterized by redness, pain, heat, swelling and loss of function.

  • Accute inflammation is an intense and short lasting inflammation. Example: response to boil.

  • Chronic inflammation is a less intense but long lasting inflammation. Example: response to tuberculosis.

Process of Inflammation (3 Stages)

1. Vasodilation and increased permeability of blood vessels.

  • Vasodilation is the dilation of blood vessels that increase blood flow to the damage tissue causing erythema (redness) and head associated inflammation.

  • Increased permeability allows fluid to move from blood into tissue spaces causing edema (swelling).

  • Histamine is a chemical substance released by damaged cells in response to injury and the causative agent of vasodilation and increased permeability.

  • Abscess is the focus of infection consisting of localized collection of pus, dead cells and body fluids in a cavity as a cause of the breakdown of body tissues.

2. Phagocyte Migration and Phagocytosis.

  • Margination is the sticking process of neutrophils and monocytes to the inner surface endothelium of blood vessels.

3. Tissue Repair

  • Stroma is the supporting connective tissue that causes scar for example in the case of major tissue damage.

  • Parenchyma is the functioning part of the tissue that can repair minor tissue damage with no visible signs.

Fever

  • Fever is an abnormally high body temperature. (above 37 deg Cel or 98.6 deg F)

  • Hypothalamus is the thermostat of the body (Microorganism releases LPS Phagocyte release interleukin-1hypothalamus releases prostaglandin reset hypothalamus thermotat)

  • Shivering and chill is a sign of that body temperature is increasing.

  • Crisis is the phase of the fever when the body temperature is falling.

Antimicrobial Substances

1. The Complement System

  • Complement system is a defensive system consisting of over 30 proteins produced by the liver and found circulating in the blood serum.

  • Proteins of the complement system destroy microbes by cytolysis, inflammation, phagocytosis, and prevent excessive damage to host tissues.

2. Interferons

  • Interferons (IFNs) are antiviral proteins and host-specific produced by certain cells such as lymphocytes and macrophages to prevent viral multiplication.

3. Transferrins

  • Transferrins are iron-binding proteins found in the blood, milk, saliva, and tears that inhibit the growth of bacteria by reducing the available amount of iron.

4. Antimicrobial Peptides

  • Consist of dozen amino acids produce my mucous membrane cells and phagocytes that binds to microbial plasma membranes to cause lysis.

Lab Report 2

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Exercise 12, page 95

2. How did the results observed on the mannitol salt and EMB correlate to the Gram

reaction of the bacteria?

Answer:

The results observed on the manitol salt and EMB correlated to the Gram reaction of

the bacteria very well. Infact, Escherichia coli and Pseudomonas aeruginosa both gram

negative, did not grow in the manitol salt agar as expected since manitol salt agar is

selective against the growth of negative organism. Whereas, Micrococcus luteus and

Staphylococcus Epidermidis which are gram negative grew in EMB, since EMB is

selective for the growth of negative microorganism.

3. Which medium is selective?

Answer:

Both medias, manitol salt and EMB are selective since mannitol salt is selective for the

growth of gram positive and EMB allows the growth of gram negative microorganism.

Exercise 13, page 102

1. Which organism(s) gave a positive test for starch hydrolysis?

How can you tell?

Answer:

The organism that gave positive test for starch hydrolysis is the Bacillus subtilis. This is due to its hydrolytic enzyme amylase. This was observed after the addition of iodine. There was an obvious clearing around areas of starch hydrolysis.

Exercise 14, page 110

2. If an organism metabolizes glucose aerobically, what result will occur in the fermentation tubes?

Answer:

Since the organism requires oxygen, aerobics and fermentation is a test in the anerobic environment or no oxygen, there will be no change.

3. Were these media differential or selective?

Answer:

The medias are differential since MRVP differentiates among family

Enterobacteriaciae based on production of acid end products from fermentation

of glucose. Citrate is differential since it differentiates members of genus Escherichia

from genera Enterobacter and Klebsiella.

Exercise 15, page 116

Critical Thinking

1. Why is agar used as a solidifying agent in culture media instead of gelatin?

Answer:

Agar is used as a solidifying agent in culture media instead of gelatin because there are few microorganism if exist that can degrade agar structurally. Whereas, gelatin can be hydrolyse by bacterias. For instance, once gelatin is hydrolyze by an exoenzyme, it liquefies and does not solidify even though when cooled below 20 deg Celsius.

Exercise 17, page 128

2. Differentiate between aerobic respiration and anaerobic respiration.

Answer:

Aerobic respiration is the release of energy from the glucose in the presence of Oxygen. The final electron acceptor is molecular oxygen (O2).

In anaerobic respiration, energy is produced from glucose without oxygen. The final electron acceptor is inorganic compounds other than O2.

Other Questions (Given by Professor)

1. Which bacteria or name bacteria can grow in 55 deg C?

Answer:

Thermophilic bacteria grow at this temperature such as Bacillus stearothermophilus.

2. Name a bacteria that can grow at room temperature.

Answer:

Mesophilic bacteria grow at this temperature such as Serratia marcescene.

3. Name a bacteria that produce red pigment?

Answer:

Bacillus subtilis

4. Name bacteria that produce green pigment

Answer:

Pseudomonas aeruginosa

Chapter 7 Lecture Slide Presentation

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