Experiment 2 & 3

1. Video:

2. Discussion:

3. Exercises:

Culture Media Preparation and Autoclaving

Bacterial cultureSterilization and disinfection

All students: Streak platesGroup 1: Examination of the bacterial distributionGroup 2: The ultraviolet radiation testGroup 3: Effectiveness of hand washing

Experiment 2

Bacterial Cultivation

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Requirements for bacterial growth

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Temperature

Nutrients

H2O, C-source, N-source, Inorganic salts, Growth factors

pH

Gas

Temperature, gas

Nutrients, pH

n

incubator

culture medium

®

®

4

n Culture medium

– is the mixture of various nutrients that is suitable for the growth of microorganisms.

Culture medium preparation and autoclaving

Video:

n Types of Culture Media•

•

based on the physical state

based on the function and chemical

components

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Based on the physical stateLiquid medium

Solid medium:

Semisolid medium:

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: Without agar.for the proliferation of bacteria.

1.5-2.5% agar.for the isolation and identification of bacteriae.g., slant, Petri dishes/plates.

0.3-0.5% agar.for the observation of bacterial motility and preservation of bacteria.

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Bacterial growth patterns

• In liquid medium For the proliferation of bacteria:

Superficial growth;

Turbidity/diffuse;

Precipitate growing;

(sediment)

§ In solid medium

Colony

For the isolation and identification of bacteria

:

Confluent growth / Smear;

: a cluster of microorganisms growing on a solid medium. It is directly visible and arises from a single cell.

Colonies of medium sized, smooth and glossy, convex, opaque, inaurate liposolubility pigment)

Colonies of bigger colonies, irregular borderline,

platode, moist, green water-soluble pigment)

S. aureus Pseudomonas aeruginosa（

金黄色葡萄球菌的菌落（中等大小、光滑、凸起、不透明、产脂溶

性金黄色色素）

（

铜绿假单胞菌的菌落（菌落大、边缘不整齐、扁平、湿润、产

水溶性绿色色素）

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§ In semi-solid medium:

A. non-motile B. motile

For observation of bacterial motility and preservation of bacteria

• Only grow along the line of inoculation

• Grow diffusely (cloudy)

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--contains the basic nutrients for most of bacterial growth; --the base of other kind of media.--e.g. broth.

Additional or special nutrients (e.g., serum, growth

factors, trace elements) are added to support some

fastidious bacterial growth.

e.g. blood agar.

Based on the function and the chemical components:

Basic Medium

Nutrient Medium/Enriched Medium

•

l

Colonies of with zone of hemolysis on a blood agar plate

White colonies of on a blood agar plate

S. aureus S. epidermidis

Staphylococcus – blood agr plates

金黄色葡萄球菌在血平板上的金黄色菌落及溶血环

表皮葡萄球菌在血平板上的白色菌落

Dewdrop-like colonies of on a chocolate plate

Neisseria meningitidis

脑膜炎双球菌在巧克力平板上的露滴状菌落

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the medium that can prevent the certain bacterialgrowth while permitting others. e.g. SS agar (Salmonella Shigella Agar)

Some special substrates and indicators are added into the media in order to produce a visual differentiation when several bacteria grow on the same kind of medium.e.g. Kligler Iron Agar

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Selective Medium

Differential Medium

Butt: semisolid, containing glucose and Ferrous ammonium sulfate

Slant: containing lactose

pH indicator: phenol red

Slant agar of Kligler Iron Agar

克氏双糖铁斜面

Bacillus proteus E.coligrowth in Kligler Iron Agar

Black coloration indicates reduction of sulfur and production of H2S

Yellow butt indicates Glucose metabolism;Clouding semisolid agar indicates motility

Red slant indicates no lactose metabolism

growth in Kligler Iron Agar

Yellow butt indicates glucose metabolism and gas formation;

Clouding semisolid agar indicates motility

Yellow slant indicates lactose metabolism

变形杆菌在双糖铁培养基上的生长情况 大肠埃希菌在双糖铁培养基上的生长情况

Salmonella typhi Shigellagrowth in Kligler Iron Agar

Yellow butt indicates glucose metabolism and no gas production;Cloudy semi-solid agar indicates motility

Red slant indicates no lactose metabolism

growth in Kligler Iron Agar

Yellow butt indicates glucose metabolism and no gas production; Clear semi-solid agar and stabbing line indicates no motility

Red slant indicates no lactose metabolism

伤寒沙门菌在双糖铁培养基上的生长情况 志贺菌在双糖铁培养基上的生长情况

Experiment 3The control of microorganisms by

physical and chemical agents

Basic terms

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Sterilization

Disinfection

Antisepsis

Asepsis

Bacteriostatic

Bactericidal

灭菌

消毒

防腐

无菌

抑菌的

杀菌的

Sterilization

Disinfection

Antisepsis

Asepsis -

灭菌

消毒

防腐

无菌

Any process, physical or chemical, that will destroy all forms of life, including bacteria, fungi, spores, and viruses.

The reduction or elimination of pathogenic microorganisms in or on materials, so they are no longer a health hazard.

Use of chemical agents on skin or other living tissue to inhibit or eliminate microbes; no sporicidal ( ) action is implied.

No living microorganisms exists.

杀芽胞的

Bacteriostatic

Bactericidal

抑菌的

杀菌的

Inhibits the growth of microorganisms

Kills microorganisms

Controlling Microorganisms By Physical Agents

High Temperature

Radiation

Filtration Low Temperature

Desiccation

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辐射

过滤

干燥

High Temperature

static action cidal action

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Dry heat Hot air sterilization

Incineration

Red heat

Flaming

Powders, Oils, Glass, etc.

干热：干烤

焚化

赤热

烧灼

protein oxidation

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Moist heat

Autoclaving:

Bacterial culture media, Operating gown, Surgical instruments, etc.

湿热：denature proteins and melt lipids; more effective

121 ,103.4kPa, 20min

cidal for both vegetative organisms and endospores

℃

Milk, Alcohol

Boiling water

Pasteurization :

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煮沸灭菌

巴氏消毒法to kill particular spoilage organisms or pathogens

flash method : 71.6 , 15s

holding method : 62.9 , 30 min

瞬间法 ℃

持续法 ℃

Radiation

Ultraviolet Radiation

Ionizing Radiation

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紫外线辐射

电离辐射

Ultraviolet Radiation•

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Microbicidal activity of ultraviolet (UV) light depends on:

length of exposure

wavelength of UV: 260 nm - 270 nm

Mechanism: thymine-thymine dimmers 胸腺嘧啶二聚体

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damage the eyes, cause burns, and cause mutation in cells of the skin

very poor penetrating power

Operating rooms; aseptic filling rooms;Surface of materials

Ionizing Radiation 电离辐射

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X-rays and gamma rays

more energy and penetrating power than UV

used to sterilize pharmaceuticals and disposable medical supplies such as syringes, surgical gloves, catheters , and sutures

used to retard spoilage in seafoods, meats, poultry, and fruits

导尿管 缝合线

Filtration

Serum, Enzyme solution, Antibiotic solution, etc.

过滤

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The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-0.45 µm (filtration membranes are most popular for this purpose).

Sterilize solutions that may be damaged or denatured by high temperatures or chemical agents

Controlling Microorganisms By Chemical Agents

DisinfectantsAntiseptics

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Commonly used Disinfectants

Classification ActionsCommonly used

DisinfectantsPractical uses

Phenol and derivatives

Alcohols

Halogens

Heavy metals

Aldehydes

Soaps and detergents

Acids and alkalies

Denature proteins, alter membrane permeability

ChlorhexidineAntiseptic for skin and mucous membranes

Denature membranes 70%~75% ethanol Skin antisepsis

Denature proteinsChorine gas iodineIodophors

Treat drinking water;Pre-op skin disinfection

Denature proteinsMercurochrome, merthiolate, Silver nitrate

Antiseptic for skin; Prevent gonococcalophthalmia

Denature proteinsFormalinglutaraldehyde

Embalming, Disinfection of endoscopes

Denature proteins, alter membrane permeability

Benzalkonium bromideHand washing, surgical hand scrub

Alter membrane permeability, denature proteins and other molecules

Undecylenic acidDermatophyteinfections

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Physical agents

Chemical agents

Temperature

Dry heat

Moist heat

Normal pressure

Autoclaving 121 , 15-20min (103.46kpa)

Pasteurization

Boiling water

Steam heating

Fractional sterilization

Ultraviolet Radiation 265 266nm most cidal, distance 30cm 1m

Filtration 0.22 µm; useless for virus, mycoplasma and some L-form bacteria

Flaming

Hot air sterilization

- Disinfectants

Ionizing Radiation

Radiation

Inactive for killing spores

℃

（ ～～ ）

Exercise: Streak-plate techniqueVideofour-area streak plate technique

Rotate

FlameRotate

Rotate

Flame

Flame

IV

II1/5 I

1/10

plate 90

loop90

90III

1/4

loop

loop

°

°

°

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Exercises

Group 1: Examination of the bacterial distribution

Group 2: The ultraviolet radiation test

Group 3: Effectiveness of hand washing

Mark your freshly inoculated plate/tubeNOTE!!!

1. The most common and effective way to kill bacterial spores:

2. What of the following is not the appropriate method insterilization?

Discussion

a. UV radiationb. Boiling for 5 minutesc. Pasteurizationd. Free flowing steam sterilizatione. Autoclaving

a. Operating room - UV radiationb. Excreta - Bleaching powderc. Water - Chlorine gasd. Culture media containing serum – Autoclavinge. Thermometers - 75% Ethanol

3. a. The effectiveness of UV light is related with its wavelength

b. UV damage DNA of microorganisms

c. UV with a wavelength of 260-270nm has strongest power to kill bacteria

d. UV has very poor penetrating power, so do no harm to human body

e. UV light is used to reduce microbial populations in air and on material surfaces

a. 100 b. 95 d. 50 e. 30

Which of the following concept on UV radiation is wrong?

4. The concentration of ethanol for disinfection is :

c. 75％ ％ ％ ％％

5. The action of which of the following agents or processes on bacteria can be reversed?

A. A disinfectant

B. A bactericidal agent

C. A bacteriostatic agent

D. Autoclaving at 121oC for 15 minutes

E. Dry heat at 160-170oC for 1 hour

1. Operating rooms; aseptic rooms; Material surface

2. Basic media; Surgical instruments

3. Milk; Alcohol

4. Serum; Antitoxin solution; Glucose solution

5. Powders; Oils; Glass

A. AutoclavingB. Ultraviolet RadiationC. PasteurizationD. FiltrationE. Dry heat

The following slides are for your reference

Factors determining usefulness of chemical disinfection

Spectrum of antimicrobial activity

The kinds and numbers of microorganisms present

Used at correct concentration

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is it the right agent for the job?

concept of 'in use concentration’

diluted down from high concentration

stored for <24 hours

no topping up of old solutions

Factors determining usefulness of chemical disinfection

Time of exposure

Correct pH?

Inactivating materials

Is disinfectant sterile?

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You cannot disinfect an endoscope in 5 minutes glutaraldehyde!

Pus, blood vomit, cork, soaps, etc.

Many cases of Gram-negatives living in disinfectants!

Microbiological “in-use” testing

Benzalkonium bromide - pH 9

Hypochlorites & chlorine

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Hypochlorites (household bleach) & chlorine

Advantagesactive against viruses, spores, fungi

Disadvantagesinactivated by organic matter, freshness & pH critical (go off if diluted), corrosive to metals

Practical Uses0.1% hypochlorite used as general disinfectantStrong hypochlorite (0.25%) used in lab & on woundsExtra strong (1%) used on HBV blood spillsChlorine used to treat drinking water and control Legionella

Iodophors & iodine

Advantages

Disadvantages

Practical Uses

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Some activity against viruses, spores, fungi

inactivated by organic matter, can stain skin, irritant, expensive

Pre-op skin disinfectionPovidone iodine used as surgical scrub, as powder on ulcers

Alcohols

Advantages

Disadvantages

Practical uses

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Isopropanol & ethanol

kill vegetative bacteria on clean surfaces in 30 seconds

inactive against spores, fungiInflammableNeed to be at correct %age with water (65-80%)

Skin antisepsis before venepunctureHand rubsDisinfection of e.g. trolley tops

Aldehydes

Advantages

Disdvantages

Practical uses

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Glutaraldehyde and Formaldehyde

Good activity against spores, virues, fungi

Glutaraldehyde only moderately active against TB

Need long exposure time for full effect (3 hours)

freshness & pH critical

TOXIC!

Disinfection of endoscopes

Blood spills

Fumigation

Ethylene oxide

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Highly toxic flammable gas, kills spores!

Used for bulky items such as heart lung machines

Can be used on glutaraldehyde-labile endoscopes

Use limited by safety issues

Phenolics & QACs

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Clear soluble phenolics (e.g. Hycolin) used as disinfectant on soiled surfaces, relatively inactive against spores and viruses

Hexachlorophene used as surgical scrub

Quaternary ammonium compounds, e.g. cetrimide usually only used in combination with other agents; good detergent properties.

Chlorhexidine ( ) 洗必泰

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Used as general purpose antiseptic for skin and mucous membranes in many formulations, e.g. Hibiscrub, Hibisol, SavlonAdvantages: relatively non-toxic and good against S. aureusDisadvantages: can support growth of e.g. P. aeruginosa