92學年度第1學期微生物學教學大綱

99學年度第2學期臺東大學

應用微生物學[Applied Microbiology]教學大綱

上課地點：生技實驗室

教師： 李炎 yenlee@nttu.edu.tw http://www1.nttu.edu.tw/yenlee/

學分數/每週時數：3/3 (前6週各上課1節實驗2節；後11週實驗為主)

評分：期末考20%、讀書報告(心得)20%、實驗成果或報告依指定方式繳交40%、實驗產品銷售體驗20%(必須研創1種應用微生物技術產品且在校內或校外擺攤銷售並取得顧客回饋表至少30張才算

(讀書: 李炎著:藍菌研究。藝軒。2005. )

實驗分組 ：1人1組，實驗產品銷售體驗5人1組

實驗室安全規則與防護用具

實驗室安全：

1. 各實驗室器材、儀器、藥品等非經實驗室負責人許可，不得擅自攜出。

2. 各實驗室應依教師規定處理化學藥品廢液、氣瓶、液態氮、及其他器材。

3. 非經實驗室負責人許可，切勿於午夜十時以後，及假日單獨進行實驗。

4. 欲使用實驗室及其設備者，須符合下列條件 :

A、確實遵守本實驗室安全規則及相關實驗室負責人之督導。

B、必須選修本系實驗技術相關課程，或接受本系一小時之實驗室安全講習。

C、接受各相關實驗室負責人之考核合格，獲准予使用該實驗室之設備者。

化學藥品

一、任何的容器都必須貼上標籤，註明其內容及有效時間。

二、任何化學藥品，都必須經過實驗負責人之許可才可以存放在實驗室中，並注意其儲藏條件。

三、在使用任何化學藥品前，一定要熟知該化學藥品之危險性。

四、在使用任何危險化學藥品時，必要時一定要穿著保護衣物，攜帶時也必須小心注意。

五、不要同時拿酸 (鹼) 與溶劑。

六、當抽風箱儲存有藥品時，一定要確定抽風箱及其內裝備使用正常。

七、打開危險化學藥品時，一定要在有保護的抽風箱內進行，並使用防濺面罩。

八、酸 (鹼) 瓶外部不得有酸 (鹼) 液殘留，要用無纖維毛巾擦拭，事後毛巾以水洗淨可丟棄。

九、實驗室內，所穿之實驗衣、手套，要定期清洗。

十、在使用完化學藥品後，要清洗你的手及所戴手套。

動物

一、欲養之動物需經指導教師同意並報戶口後，方可飼養，並嚴禁未經許可而自行飼養。

二、請維持整齊與清潔，所有物品使用後請歸回原位。

三、動物屍體請包裝完整，置於特定冷凍冰箱暫時儲存，以便集中送焚化，切勿丟在垃圾場，影響環境衛生。

廢料

一、化學藥品

A、使用完之空瓶處理

1、酸 (鹼) 瓶: 已使用之空瓶，先在排酸 (鹼) 之水槽將空瓶以水清洗乾淨，才可丟棄。

2、溶劑瓶 : 已使用完之無污染性溶劑，放在溶劑之抽風箱內，至少抽氣四小時才可丟棄。

3、用完藥品 (有污染性) 之空瓶蓋須蓋緊集中於一處，待一定量時送交校內相關環保單位處理。

B、有機溶液

1、千萬不可將使用過之溶劑，再倒回原來之容器內。

2、請使用有機溶液桶，若不合規定，使用其他裝置容器，待一定量時送交校內相關環保單位處理。

3、有機溶液桶裝八分滿即止，切勿裝過多。

4、儲放廢棄溶劑桶處，一定要具有通風設備。

5、三氯乙烯及任何含氯溶劑，如含多氯聯苯廢棄物，請放在專為含氯溶劑的容器中，其他溶劑則收藏在不含氯容器中。

7、廢棄溶劑之收集，將由學校集中處理。

三、培養後細胞與病毒

培養後細胞與病毒，務請使用高壓消毒滅菌鍋滅菌後，同一般垃圾丟至垃圾場。

四、玻璃

碎玻璃 (含碎玻璃、燈管、玻璃針筒)應妥善包裝，未破玻璃瓶內不能含有機溶液或化學藥品。

五、針頭

應先燒紅剪斷後才可送出。

(六) 藥品溢潑處理

一、化學藥品

任何藥品溢潑出來，隨時都會發生危險，清理工作要馬上進行，不可拖延。

1 、在抽氣櫃內:

( 1 ) 溶劑

a、避免點火及可引起火花之任何動作。

b、用無纖維之毛巾擦拭溢出溶劑，將要棄置之毛巾裝入塑膠袋中，且綁緊袋口棄置之。

( 2 ) 酸

a、在工作臺處抽氣。

b、噴灑去離子水。

c 、將離子水抽乾，擦乾。

d 、重複b．和c．。

e、將所用之毛巾清洗及塑膠袋綁緊，再棄置。．

2．抽氣櫃外

( 1 ) 溶劑

a、避免點火及可引起火花之任何動作。

b、去最近的地方，拿噴灑吸收溶劑之乾粉，將噴灑吸收劑由外而內灑濺有溶劑處。

c、用剷子將吸收劑清理掉。

( 2 ) 酸和鹼

a、去最近的地方，取中和酸(鹼)劑，由外向內噴灑處，用試紙測試是否還在處。

b、將中和劑清理掉。

c、用肥皂及水清理濺灑處。

( 3 ) 有危險揮發性藥品在抽風櫃外濺出，且超過30ml時：

a、撤退此區人員，且幫助被濺到的人，通知安全人員，實驗室負責人。

b、在專門人員清理前莫入實驗室。

(七) 意外事件處理

一、發生可控制的火災時:

1、使用附近的滅火器 :

a、揭開環狀保險栓。

b 、擠壓槓桿。

c、將噴嘴對火苗底部。

2、若是衣服著火，用滅火毯掩蓋它，以達到窒息火苗的目的。

3、若是電線走火，應趕快關閉電源。

4 、迅速向實驗室負責老師報告。

二、當發生無法控制的火災時 :

1、將人員、物資撤離火災現場。、

2、離開實驗室時，將所有電源關掉。

3、打電話通知校園警衛及院、系、館、負責人赴援。

三、當發生嚴重意外，傷者需要立即送醫時

1、打電話通知校園警衛及校護。

2、若是可能，應依照下面步驟給予傷者初步的幫助。

a 、若必須自危險地區搬離傷者，則小心勿使他 (她) 的頭受到傷害。抬他 (她) 至安全地方，保持安靜，並儘量使傷者舒服一點。

b、若是傷者的傷勢容許的話，應召喚並等候一位接受過訓練的人，讓他來做初步的幫助。

3、若是傷者的傷勢嚴重，當等候救護人員時可做下列步驟 :

a、當傷者皮膚被化學物所傷時 :

( 1 ) 除去被化學物污染的傷者衣服。

( 2 ) 用清水沖洗傷部至少十五分鐘。

( 3 ) 給予傷者醫藥處理，並召喚救護人員。

b、當傷者眼睛被化學物所傷時 :

( 1 ) 用洗眼液洗滌眼睛至少十五分鐘，不時地拉起傷者上下眼皮。

( 2 ) 請人幫忙，將傷者立即送醫診治，不管傷者眼睛感覺如何。

4 、傷者被毒氣中毒時 :

a 、找出原因並處理。

b、將傷者抬至通風處，鬆解衣扣，領帶等束縛物。不要嚐試去脫傷者的衣服。

四、如何打電話通知校園警衛及校護等

1、撥校園電話分機：校護1220、生命科學系辦公室6500。

]2、報告自己姓名。

3、陳述火災或警急意外事故發生地點。

4 、報上自己所用電話的號碼。

5、儘可能明白清楚地陳述事件的發生原因。

6 、除非對方先掛上電話，切勿先掛電話。

切結書

本人，已確實研讀臺東大學生命科學系實驗室安全規則，願意遵守該項規則之各項規定，並接受實驗室負責人之督導考核。如因違反規則或疏忽，而造成之傷害損失，本人願負全責。

切結人(簽名)：

教師：

中華民國年月日

請簽名後撕下本頁交給教師

進度：

!st week：應用微生物學概述

部份內容Modified from: Kathy Huschle

Northland Community and Technical College

The scope of Microbiology：

bacteria [cyanobacteria], viruses, fungi , protozoa, algae, helminths, prions,

The Impact of Microbes on Earth:

Microbial involvement in energy and nutrient flow, Photosynthesis, decomposition,

Applied microbiology is the interaction of the microbial world and the rest of the world

-genetic variances

-microbial effect on soil, water, environment, our food

Microorganisms are present in most every aspect of our lives

Microorganisms are critical to our survival on Earth

Microbial Ecology:
-relationship of microorganisms with each other and their environment

Ecosystem: interaction of living and non-living components

-oceans, deserts, marshes沼澤 , forests, tundra凍原 , lakes

-microorganisms play a key role in ecosystem structure

Microbial Ecology:
-relationship of microorganisms with each other and their environment

Microenvironment: immediately surrounds a microorganism

-relevant to survival and growth of the microorganism

Nutrient Acquisition within an Ecosystem：

3 main levels exist in every ecosystem in regards to nutrient acquisition

– producer

– consumer

– decomposer

1. primary producers:

convert CO2 to organic material

2. consumers

– heterotrophs

– utilize organic material created by producers

3. decomposers

– heterotrophs

– digest leftovers of primary producers and consumers

detritus風化物；殘渣；腐殖質 ( fresh or partially decomposed organic matter)

– bacteria and fungi are key players in the process of decomposition

– Bacteria do best in biofilms if nutrition availability is low

– biofilms are a polysaccharide encased community of microorganisms

– microorganisms extract nutrients that are absorbed by water from air or nutrients that are adsorbed onto the biofilm

Competition and Antagonism: among microorganisms

Competition:

– fierce competition for nutrients and water

– the faster a microbe reproduces the larger the population

– the larger population competes better

critical, especially if the microorganisms competing utilize similar nutrients

Antagonism

– bacteriocins: protein produced by bacteria that destroys similar strains

“WINNER TAKES ALL AND IS KING/QUEEN OF THE MICROBIAL ECOSYSTEM”

Stability of microbial community in human intestine is attributed to competition and antagonism amongst its members

– compete nicely for nutrients

– produce toxins to limit growth of new microbes

Environmental Change affect microbial population

Environmental fluctuations are common and resident microorganisms may respond by

– producing enzymes to help adapt to changing environment

additional or different enzymes may be necessary for survival

– mutation

– domination by other species (can’t compete any more)

Microbial Mat:
thick, dense, organized biofilm

generally found attached to a solid substrate or at air-water interfaces

The Study of Microbial Ecology

Somewhat difficult to accomplish

– less than 1% of environmental microorganisms can be successfully cultured in the lab

Microbial Habitat

aquatic

– marine:

deep waters are usually stable and consistent

shoreline habitat varies due to nutrient rich run-off

freshwater:

– lakes

stratification allows for the mixing of the water seasonally.

Increases the presence of O2 in the deeper H2O

moving water

– rivers

generally aerobic due to turbulence facilitating幫助 O2 circulation

terrestrial

– microorganisms are critical to soil habitat

composition of microbes is dependent on soil conditions

wet soil: anaerobic conditions due to water filling the pore space in the soil, soil dries and microbes go produce endospores for survival

Mutualism互利共生 with Eukaryotes

mychorrhizae菌根 : fungus

– assist plants in the uptake of phosphorous

– mychorrizae gain nutrient from plant

nitrogen fixers: fix nitrogen and make it available for the use by their partner plant

– most common is Rhizobium, a microorganism found in many root nodules

microorganisms and herbivores

– animal with a rumens (cow) or cecums盲腸(horse) need microorganisms to digest the plant food they ingest

Microorganisms in Sewage Treatment

decreasing biochemical oxygen demand (BOD) decreases impact of sewage on the environment

BOD is the amount of O2 needed for microbial decomposition of the organic material in a sample

if not treated the high BOD found in sewage could deplete the O2 level in the receiving water

in other words if raw sewage is deposited into a lake or stream without treatment, it would effectively suck the oxygen out of the water, leaving very little for the fish and other organisms

sewage treatment is a controlled process that strives to eliminate the excess organic material, thus diminishing the BOD

most of the removal of organic matter is done by microorganisms

municipal water supplies are tested and treated for the removal of pathogenic microorganisms and chemicals

this is done with the use of chemicals

the elimination of organic waste material can be enhanced by microorganisms

increase cost to separate organic material from inorganic (glass, metal, plastic)

composting: natural decomposition of organic solid materiel results in excellent fertilizer

Compost

Bioremediation:
use of microorganisms to eliminate or make harmless pollutants in an environment

pollutants removed can include

– organic solvents

– toxic chemicals

– hydrocarbons

introduces specific organisms to the polluted area

– many toxic substances are man-made/new to the environment (xenobiotics)

– no time for naturally occurring microbes to have evolved biochemical pathways for their degradation

scientists are trying to develop new microbes for the degradation of environmental polluters

scientists are also making use of organisms already found in the environment

– enhance their requirements for growth, such as nutrition or water availability

current methods of controlling some environmental polluters are incineration or storage in land fills, which result in

– more pollution

– health risks

bioremediation is

– inexpensive

– publicly accepted

– non-polluting (ideally)

– in situ treatment (at the site)

Food Microbiology

food is an ecosystem and microorganisms play a key role in the stability of that ecosystem

microorganisms are introduced to the food ecosystem from the soil, harvesting, handling, storage, and packaging

fermentation: good food microbiology

food that have been intentionally altered such as sour cream, cheese, beer

any desirable change a microorganism makes to food

spoilage: bad food microbiology

undesirable changes to food; sour milk, moldy bread

preservatives and refrigeration inhibit the growth of microorganisms

factors that affect the presence of microorganisms in food include

intrinsic

extrinsic

Intrinsic growth factors:
naturally present in food

water availability is measured as water activity (aw)

this is the amount available in the food

most microorganisms require an aw of 0.90 or above for growth

fungi can grow with a aw of 0.80

fresh food have an aw 0.98

Definition of aw:

aw=P/Po

where p is the vapor pressure of water in the substance, and p₀ is the vapor pressure of pure water at the same temperature.

Intrinsic Growth Factors

– many species of bacteria are inhibited by low pH, including most pathogens

biological barriers: shells, rinds外皮

– protect foods from invading microorganisms

antimicrobial chemicals:

– naturally occurring in some foods

egg whites have lysozyme which will destroy lysozyme susceptible bacteria

Extrinsic Factors:
environmental conditions

temperature of storage

– below freezing water is unavailable for microorganisms

– low temperatures (above freezing) enzyme reactions are non-existent or slow

– refrigerated food microbial growth is likely psychrophiles

atmosphere: presence or absence of O2

– obligate aerobes (need O2) won’t grow in sealed containers

may allow growth of anaerobic microbes

Microorganisms in Food Production

using microorganisms for food production has been done for thousands of years

– cheese, yeast, beer

microorganisms used in food often produce an acidic by-product as a result of metabolism

– can inhibit growth of many spoilage microorganisms

– can inhibit growth of many foodborne pathogens

Food Spoilage:
undesirable changes in food

smell bad, taste bad, look bad

probably are not harmful

– microorganisms that cause food spoilage compete with pathogens

– in the case of food spoilage vs. pathogens, the spoilers are winning

evidence is obvious, though I wouldn’t eat anything that smelled or looked like that

Foodborne Intoxication

illness from microbial exotoxin

– microorganism does not cause the illness, the toxin released by the microorganism does

common exotoxin producing microorganisms

– Staphylococcus aureus

– Clostridium botulinum

Foodborne Infection

requires consumption of microorganism

symptomatic about 1 day following ingestion of contaminated food

common foodborne infecting microorganisms

– Salmonella and Campylobacter

poultry product infections

– Escherichia coli 0157:H7

undercooked hamburger

Food Preservation:
preventing growth and metabolic activities of microorganisms

spices, salting, drying are methods that have been around for years

most common methods of current food preservation are

– high temperature treatment

– low-temperature storage

– antimicrobial chemicals

– irradiation

Human Use of Microorganisms:

bioremediation, medicine [antibiotics] , industry, food, bio-renewable energy production, genetic engineering , ….

Classifying Living Things

ARCHAEA: Extremophiles

Extremophles (X-trem’-o-files)

Require extreme conditions of temperature, salinity or pH to survive.

Largest group of Archaea.

Produce methane as a metabolic byproduct.

Common in wetlands (responsible for marsh gas)

In the guts of animals such as ruminants and humans (where they are responsible for flatulence)

Bacteria

Gram-Negative & Gram-Positive

Mycobacterial Cell Wall

Because of waxy cell wall, they can survive exposure to acids, alkalis, detergents, oxidative bursts, lysis by immune system, and many antibiotics.

Lactose Fermentation

用麥康基培養基

Chlamydia trachomatis

All bacteria are prokaryotes

Eukaryotic Cell Structure

endomembrane system

Protozoan Life Cycle

Fungi:

http://www.ied.edu.hk/has/bio/dlo/fungi/fun.htm

http://www.tolweb.org/Fungi

http://www.perspective.com/nature/fungi/

http://calphotos.berkeley.edu/fungi/

http://fungi.fvlmedia.dk/

http://www.personal.u-net.com/~chilton/fungi.htm

http://www.first-nature.com/fungi/index.htm

Virus:

The Reproductive Cycle of a Retrovirus

Modified “Live” Virus Vaccines vs “Killed” Viruses Vaccines

Modified live virus vaccines contain viruses that have been altered (attenuated) to virulence, yet retain their antigenic properties and induce a immune response.

MLV vaccines must replicate after inoculation to produce enough antigen to produce an immune response.

Advantages:

- One dose

- Quicker immune response

- Stronger, more durable response

- Fewer post-vaccine reactions

Killed virus vaccines contain viruses that have been treated by chemical or physical means to prevent them from replicating in the vaccinate.

Advantages:

- Safer

- No possibility for reversion

- Recommended for pregnant animals

- Stable in storage

Prion:

Basic Chemical Reactions Underlying Metabolism

Oxidation and Reduction Reactions

ATP Production and Energy Storage

Anabolic Reaction

(anabolism)

Catabolic Reaction

(catabolism)

Carbohydrate Catabolism

Glycolysis

Fermentation

API-20E

Microbial growth

Factors Influencing Microbial Growth

• Nutritional requirements

• Oxygen requirements

• Temperature

• pH

• Osmotic Pressure

http://www.mcb.uct.ac.za/tutorial/classif.htm

http://en.wikipedia.org/wiki/Virus

http://web.uct.ac.za/depts/mmi/stannard/emimages.html

Protozoa;

http://www.nies.go.jp/chiiki1/protoz/

http://zh.wikipedia.org/wiki/%E5%8E%9F%E7%94%9F%E5%8A%A8%E7%89%A9%E9%97%A8

http://content.edu.tw/junior/bio/tc_wc/cairoom/8711/TOP/1.htm

http://content.edu.tw/junior/bio/tc_wc/textbook/ch10/supply10-3-0.htm

http://www.pirx.com/droplet/

http://micro.magnet.fsu.edu/moviegallery/pondscum/protozoa/amoeba/28k/amoeba01.html

Lab Report:[Introductio, Materials and Methods, Results, discussions, References]

Experiment: 校園水池藍菌分離檢測

[在4週內繳]

2nd week:

Culturing microbes：

http://www.mansfield.ohio-state.edu/~sabedon/biol4035.htm

Methods used in bacterial identification:

Microscopic morphology

Physiological /Biochemical characteristics

Chemical analysis

Serological analysis

Genetic and molecular analysis

醱酵作用（以有機物為最終電子接受者）：

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookGlyc.html

光合作用菌：

http://www.jochemnet.de/fiu/bot4404/BOT4404_12.html

純菌分離技術（畫線、L型玻捧製作與塗碟、倒皿、稀釋）、pipetter使用、接種於Agar slant、無氧培養法（燭箱、油隔）、

細菌計數（稀釋塗皿—E.coli與Nocardia yen—N.yen、光譜）、光譜儀使用、Coulter counter使用、

純菌真空乾燥保存技術、冷凍保存技術、黴菌培養、

食物中菌數測定、自來水中菌數測定、

黴菌培養結果觀測、細菌分類API 20E試條使用

每位同學送1株純化分離的cyanobacterium(以13 x 100mm BG-11 agar slant 保存,附細菌顯微照片--100x接物鏡頭)至老師處評分--10%

各種病菌：

http://www.merck.com/mrkshared/mmanual_home2/fg/fg190_1.jsp

http://textbookofbacteriology.net/medical.html

檢測：

Thayer-Martin medium[淋病]

Lowenstein-Jensen medium[Mycobacterium]

EMB

Stainer-Scholte medium

Brilliant green medium

TSI

原理為 TSI 為斜面培養基含三種醣（glucose, lactose, sucrose），並加入硫化亞鐵及phenol red指示劑；因細菌對醣類發酵反應的不同，利用氣體的產生，硫化氫的形成來鑑定革蘭氏陰性桿菌。

http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Triple_Sugar_Iron/TSI_Use.htm

SIM

SIM（sulfide-Indole-Motility medium）：檢查細菌分解色氨酸（L-Tryptophan）而產生Indole與還原硫的能力。

MR-VP

IMViC

TCBS

http://microbiology.scu.edu.tw/wong/research/vp.htm

Hemagglutination inhibition

ELISA

http://www.biology.arizona.edu/immunology/activities/elisa/technique.html

3rd,4th,week:

環境微生物

Lab ：

Isolation of petroleum-degrading bacteria：

（Biodegradation pathways：http://umbbd.msi.umn.edu/ ）

Purpose- To determine the relationship between soil environments and the activity of petroleum-degrading bacteria.

Background information-Petroleum is a rich source of organic matter. It is not surprising, therefore, that a wide variety of microorganisms will readily attack it under certain environmental conditions. This demonstrates a beneficial role of microorganisms- the biodegradation of pollutants. Significant breakdown will only take place in the presence of oxygen. If the oil is deposited into an anaerobic environment (absence of oxygen), decomposition will not occur and it may remain in place for many years. This helps to explain why natural oil deposits may be millions of years old. The physical properties of oil, which are insolubility in water and low density, will explain the formation of oil slicks（水面）. The oil is exposed to oxygen and is quickly attacked by oil-degrading bacteria, which eventually decompose the oil and disperse it. Petroleum degraders include a variety of bacteria, certain molds and yeasts. Most soil should have bacteria, capable of using oil. Motorcycle service stations most often have oil-soaked soil, which is an excellent source of these microorganisms. Oil-degrading bacteria often use other common organic chemicals, such as moth（樟腦） balls, as nutrients.

Materials
5-clear, covered experimental containers (125ml minimum)
5- volume-measuring containers
4- 10 ml. Pipettes and pipette pumps
4 collection bottles

1/10x Lee’s medium（gram/liter dH₂O：

MgSO₄.7H₂O 0.05g， K₂HPO₄ 0.05g， Ca（NO₃）₂.H₂O 0.05g，Na₂-EDTA 0.001g， Fe（SO₄）₃.6H₂O 0.01g，NaCl 0.05g，and add1mlmicroelements solution:

H₃BO₃ 2.86， MnCl₂.4 H₂O 1.81， ZnSO₄.7H₂O 0.222， MoO₃(85%) 0.018， CuSO₄.5H₂O 0.079， CoCl₂.6H₂O 0.01，use10% NaOHto adjust pH to 8.2，then autoclave it for sterilization。）

1/10x Lee’s medium oil plate: 1.5% agar in Lee’s medium and add 10ml/liter used mtotor oil, autoclave, and pour plates

L shaped glass rods

95% alcohol

Glass Petri dishes

Gram stain materials

Glass slides

Microscope

Kimwipes

MicroScan AS4 automatic microbe analyzer（for biochem tests）
Clear metric ruler
Distilled water
Moth balls
30 ml. Nutrient broth, or dissolved chicken bouillon（肉湯）cube
Rubber gloves
Lysol or 10% bleach solution
Paper towels

Procedure for Detecting the Presence of petroleum-degrading Bacteria

Field Activity

Collect soil samples from three different locations. Look for areas that vary in their exposure to oil. (Examples include soil near a motorcycle service station and from a garden.)
Label and date each collection bottle.

Back at the lab

Prepare an aseptic work area.
Prepare experimental and control specimens, using clean experimental containers.
Add 50 ml. Distilled water to each container. Close each container with a cover.
Using a pipette, measure 5 ml of nutrient solution. Add it to the first container and replace the cover. Repeat for each of the remaining two containers. Replace covers.
Add a 10 ml soil sample to the container with the matching label. Replace covers. Gently swirl each covered container.
Place a moth ball in each container and replace covers..
Incubate at room temperature in a space designated by the teacher for a minimum of seven days.
Without removing the moth ball, measure the vertical and horizontal axes of each moth ball, once a day for seven days.
Isolate and identify microbes from cultures.（spread bacteria 【100ul each】from each container onto Lee’s medium-oil plate separately, incubate in 37℃ incubator for 1 week, pick each different colonies, check the bacteria under the microscope, Gram stain the bacteria, re-check it under the microscope, grow the isolated pure cultures in lee’s medium with oil separately, 7days, try to identify the bacteria ）

Writing the Report

Report is to be written in standard lab report form.

Critical thinking questions (to be included in conclusion)

Describe any non-quantifiable observations that occurred in the specimens.
Describe another sampling technique that could be used to test for petroleum-degrading bacteria.
Describe any sources of error that may have affected the results.
Compare data with other groups. Describe and explain any similarities and differences you have observed.

Extensions

Survey local service stations to determine their methods of oil disposal.
Do an internet search on applications of bioremediation（You have to write your finding in the report.）.

Extract 16S rRNAs for each bacterium and identify the bacteria (Advanced—we do not do this procedure in this lab)

10%

投稿:

http://www.dyu.edu.tw/~journal/setjournal/setjournal.htm

http://www.asia.edu.tw/ajhis/

http://wwwdb.tesri.gov.tw/protect/UpLoadPic/08111348/08111348.pdf

http://www.thing.net/~grist/homecyan.htm

5-9 th week:

Lab: Isolate teeth protection phages.

Lab: Isolate antibiotics excrete bacteria.

Design a close system that contains protozoa, cyanobacteria, plant, fish, insect, .....and the closed ecosystem can survive for at least one month.

Or 醬油的釀製

v 醬油：以黃豆(或脫脂黃豆)和小麥為原料，經製麴後，與食鹽水下缸，經長時間之發酵、熟成，產生氨基酸、有機鹽、有機酸、各種糖類、醇類等特殊風味的製品。

v 發酵：凡利用微生物所分泌之酵素，使有機物發生氧化、還原或分解、合成反應者，俱可稱之為發酵。

v 原料：

v 黃豆或脫脂黃豆粉

v 小麥粉

v 麴種(Aspergillus oraze)

v 食鹽

v 水

1.黃豆蒸煮：黃豆100kg→置於罐→加水浸漬5~6小時→排水(最後以蒸氣壓出之) →蒸煮(先排氣5分鐘，升壓達1kg，一直迴轉保持25分鐘後再排氣) →真空冷卻(760mmHg，60分鐘，每隔5分鐘回轉一次)使品溫達40℃。

2.小麥焙炒與粉碎：小麥100kg→焙炒→粉碎(30mesh)→麥粉約85~90kg。

3.混合：種麴350~400g先與麥粉混合後再與黃豆混合。

4.入麴室培麴：分四段溫控培麴，其中第18~20hr，及第26hr，須翻麴。

0~10hr，風溫26~27 ℃。 10~20hr，風溫30~31 ℃。

20~26hr，風溫29~30 ℃。 26~44hr，風溫25~26 ℃。

5.出麴。

6.加22.5~23%(Be’18.5)之食鹽水360L下缸。

7.發酵熟成：至少半年，其間應攪拌，二個月內每7天一次，二個月後15天一次，且pH保持在5天以上，NaCl保持在17%以上。

8.醬120lkg加氨基酸液20kg壓榨得生醬油。

9.依比例調味：生醬油-100；砂糖-3；甘貝素-0.04；焦糖-0.4；甘草粉-0.1；味精-1；核苷酸-0.06；水-30。

10.煮沸。

11.離心過濾。

12.裝瓶。

13.封蓋。

10 %

10-13 th week:

Select functional microbes' mutants or functional microbes 10%

四川泡菜的做法

 工具：陶做的專門的泡菜罎子，壇口突起，壇口周圍有一圈凹形託盤(即水槽，可盛水)，扣上扣碗可以密封的罎子，它可以使泡菜在缺氧的情況下加速發酵，產生大量乳酸。做泡菜時要在旁邊託盤內裝滿水密封罎子，可防止空氣進入。如沒有泡菜壇，也可用別的容器代替，但要求容器口大而密封嚴密，不能透氣。現在流行一種玻璃罎子，這種罎子比較好看，從外面可以看到裏面各種顏色的菜。這也作為餐廳裏攫取顧客眼球的一種手段。

四川泡菜通常分兩種。種類不同，用途不同，做法也大不相同。

  一種是跳水泡菜，跳水泡菜的來由是因為這菜泡在罎子裏很快就取出來吃了。可以作為一個專門的菜。通常現在川菜館裏的什錦泡菜和飯前桌子上的小菜都是這類。

  材料：通常用瓜類的蔬菜或者質地堅硬的根、莖、葉、果等，切成長條。

  作法：將清水燒開，加食鹽(每1公斤水約50--60克鹽)，待鹽完全溶解後，放人適量配料，倒人泡菜壇中(以鹵水淹到罎子的3／5為宜)。待鹵水完全冷卻後，再放入菜塊。取食泡菜要使用專筷，切不可帶油，避免油與生水進入壇中，否則會生花(水表面會生白色黴菌)。壇口水槽要保持清潔，並經常換水注滿。

  吃法：萵筍，蘿蔔之類的蔬菜只需要泡八個小時至十二個小時，這樣泡出來的蔬菜脆生生的，帶點適量的鹹味，很是爽口。如果喜歡吃辣的，可以澆上點辣椒油，拌上少許味精，是難得的美味。

  另一種是做佐料用的。川菜裏有很多有名的菜式都是用這種泡菜做的，這種泡菜如果想做專門的菜，那可以比跳水鹹菜多泡制幾天，差不多一周至十天即可，比跳水鹹菜通常多一點酸味；另外一種就是長期泡在罎子裏，只在做菜的時候撈起來做為佐料。

  材料：辣椒，青菜，白蘿蔔，豇豆，大蒜、葱、酒、醋、鹽、糖、薑、花椒，八角、茴香。

  做法：

  1.將要泡的菜洗淨風乾。

  2.將清水燒開，放入適量鹽(每1公斤水約80克鹽)放在旁邊備用。

  3.起壇水：用以前的泡菜罎子裏的“母水”，或者可以向家裏已有泡菜罎子的朋友要一些，放在新鮮壇水裏，會有更好的味道，裏面有大量的乳酸菌。如果找不到就只好自己重新製作了。將已經冷卻的清水倒入母水之中。

  4.加入佐料，花椒，茴香，白酒，辣椒，青菜，白蘿蔔，豇豆，大蒜、葱、酒、醋、鹽、糖、薑、…壇水就製成了。

  5.將準備好的蔬菜入壇醃制。菜要裝滿，儘量少留空隙，以液面靠近壇口，鹽水淹沒蔬菜為宜。在壇口周圍水槽中注入涼開水，扣上扣碗，放在陰涼處。

  6.泡菜放的地方注意陰涼，注意保持壇口始終有水以保證壇中不進空氣和細菌。如發現壇中有生花的現象，加入少許白酒即可。

  備註：做好的泡菜如食用時不適口味，還可以作些調整：不脆可以加點酒；太酸可以加些鹽；發黴變味，則是壇中熱氣太高，或取用工具不潔所引起，此時應將黴點去掉，加入食鹽和少量白酒，移放陰涼處，每天敞口10分鐘左右，約3～5天黴味自然消失。如發現泡菜軟爛發臭，說明泡菜已變質，不能食用，菜鹵也不能再用。

經驗:

1 初做鹵水時要加些紅糖和四川大紅袍花椒和鹽水一起煮.紅糖即可起消毒又可產酸.花椒可起麻香的味道.

2 第一次泡時最好要泡些心裏美蘿蔔,因為它可以泡出漂亮的紅顏色,再泡點芹菜(芹菜可以產生特殊的香味)紅綠非常好看!

3 還應放些蒜.

参考资料： http://www.wsmmz.net/Archive_view.asp?boardID=31&ID=5574

臭豆腐http://carrie2008.pixnet.net/blog/post/25377447

臭豆腐滷汁:

1大匙(15ml) 蝦醬 + 350~400cc溫水 + 1/4塊嫩豆腐 + 少許鹽, 攪拌均勻, 放置室溫2天後即可使用
( 將1/4塊嫩豆腐切小小塊放入 )

做法:

將firm豆腐放到滷汁裡醃6~8小時,讓它入味

**一般豆腐太軟,泡在臭豆腐滷汁容易破掉, 建議用firm豆腐喔!~~**

**醃的時間請自己斟酌,因為天氣熱的話豆腐容易酸掉唷!~~~**

**醃過豆腐的滷汁放冰箱冷藏可以重覆使用**

藍菌飲或食品製作

14-17 Week:

創新產品實銷體驗

18th week:

Final Exam