2.5: Differential Staining Techniques - Biology

2.5: Differential Staining Techniques - Biology

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Viewing Bacterial Cells

The microscope is a very important tool in microbiology, but there are limitations when it comes to using one to observe cells in general and bacterial cells in particular. Contrast, however, can be improved by either using a different type of optical system, such as phase contrast or a differential interference contrast microscope, or by staining the cells (or the background) with a chromogenic dye that not only adds contrast, but gives them a color as well.

There are many different stains and staining procedures used in microbiology. Some involve a single stain and just a few steps, while others use multiple stains and a more complicated procedure. Before you can begin the staining procedure, the cells have to be mounted (smeared) and fixed onto a glass slide.

A bacterial smear is simply that—a small amount of culture spread in a very thin film on the surface of the slide. To prevent the bacteria from washing away during the staining steps, the smear may be chemically or physically “fixed” to the surface of the slide. Heat fixing is an easy and efficient method, and is accomplished by passing the slide briefly through the flame of a Bunsen burner, which causes the biological material to become more or less permanently affixed to the glass surface.

Heat fixed smears are ready for staining. In a simple stain, dyes that are either attracted by charge (a cationic dye such as methylene blue or crystal violet) or repelled by charge (an anionic dye such as eosin or India ink) are added to the smear. Cationic dyes bind the bacterial cells which can be easily observed against the bright background. Anionic dyes are repelled by the cells, and therefore the cells are bright against the stained background. See Figures 1 and 2 for examples of both.

Figure 1. Negative stain of Cyptococcus neoformans, an encapsulated yeast

Figure 2. Positive stain of Staphylococcus aureus.

Probably the most important feature made obvious when you stain bacterial cells is their cellular morphology (not to be confused with colonial morphology, which is the appearance of bacterial colonies on an agar plate). Most heterotrophic and culturable bacteria come in a few basic shapes: spherical cells (coccus/cocci), rod-shaped cells (bacillus/bacilli), or rod-shaped cells with bends or twists (vibrios and spirilla, respectively). There is greater diversity of shapes among Archaea and other bacteria found in ecosystems other than the human body.

Often bacteria create specific arrangements of cells, which form as a result of binary fission by the bacteria as they reproduce. Arrangements are particularly obvious with non-motile bacteria, because the cells tend to stay together after the fission process is complete. Both the shape and arrangement of cells are characteristics that can be used to distinguish among bacteria. The most commonly encountered bacterial shapes (cocci and bacilli) and their possible arrangements are shown in Figures 3 and 4.

Figure 3. Possible bacterial cell arrangements for cocci

Figure 4. Possible bacteria cell arrangements for bacilli

Differential Staining Techniques

In microbiology, differential staining techniques are used more often than simple stains as a means of gathering information about bacteria. Differential staining methods, which typically require more than one stain and several steps, are referred to as such because they permit the differentiation of cell types or cell structures. The most important of these is the Gram stain. Other differential staining methods include the endospore stain (to identify endospore-forming bacteria), the acid-fast stain (to discriminate Mycobacterium species from other bacteria), a metachromatic stain to identify phosphate storage granules, and the capsule stain (to identify encapsulated bacteria). We will be performing the Gram stain and endospore staining procedures in lab, and view prepared slides that highlight some of the other cellular structures present in some bacteria.

HomeGram Stain

Figure 5. Bacteria stained with Gram stain.

In 1884, physician Hans Christian Gram was studying the etiology (cause) of respiratory diseases such as pneumonia. He developed a staining procedure that allowed him to identify a bacterium in lung tissue taken from deceased patients as the etiologic agent of a fatal type of pneumonia. Although it did little in the way of treatment for the disease, the Gram stain method made it much easier to diagnose the cause of a person’s death at autopsy. Today we use Gram’s staining techniques to aid in the identification of bacteria, beginning with a preliminary classification into one of two groups: Gram positive or Gram negative.

The differential nature of the Gram stain is based on the ability of some bacterial cells to retain a primary stain (crystal violet) by resisting a decolorization process. Gram staining involves four steps. First cells are stained with crystal violet, followed by the addition of a setting agent for the stain (iodine). Then alcohol is applied, which selectively removes the stain from only the Gram negative cells. Finally, a secondary stain, safranin, is added, which counterstains the decolorized cells pink.

Although Gram didn’t know it at the time, the main difference between these two types of bacterial cells is their cell walls. Gram negative cell walls have an outer membrane (also called the envelope) that dissolves during the alcohol wash. This permits the crystal violet dye to escape. Only the decolorized cells take up the pink dye safranin, which explains the difference in color between the two types of cells. At the conclusion of the Gram stain procedure, Gram positive cells appear purple, and Gram negative cells appear pink.

When you interpret a Gram stained smear, you should also describe the morphology (shape) of the cells, and their arrangement. In Figure 5, there are two distinct types of bacteria, distinguishable by Gram stain reaction, and also by their shape and arrangement. Below, describe these characteristics for both bacteria:

Gram positive bacterium:Gram negative bacterium:

HomeAcid Fast Stain

Some bacteria produce the waxy substance mycolic acid when they construct their cell walls. Mycolic acid acts as a barrier, protecting the cells from dehydrating, as well as from phagocytosis by immune system cells in a host. This waxy barrier also prevents stains from penetrating the cell, which is why the Gram stain does not work with mycobacteria such as Mycobacterium, which are pathogens of humans and animals. For these bacteria, the acidfast staining technique is used.

Figure 6. Acid-fast bacilli in sputum

To perform the acid-fast stain, a heat-fixed smear is flooded with the primary stain carbol fuchsin, while the slide is heated over a steaming water bath. The heat “melts” the waxy cell wall and permits the absorption of the dye by the cells. Then the slide is allowed to cool and a solution of acid and alcohol is added as a decolorizer. Cells that are “acid-fast” because of the mycolic acid in their cell wall resist decolorization and retain the primary stain. All other cell types will be decolorized. Methylene blue is then used as a counterstain. In the end, acid-fast bacteria (AFB) will be stained a bright pink color, and all other cell types will appear blue.

Staining Methods to Highlight Specific Cell Structures

Capsule: The polysaccharide goo that surrounds some species of bacteria and a few types of eukaryotic microbes is best visualized when the cells are negative stained. In this method, the bacteria are first mixed with the stain, and then a drop of the mixture is spread across the surface of a slide in the thin film. With this method, capsules appear as a clear layer around the bacterial cells, with the background stained dark.

Metachromatic granules or other intracytoplasmic bodies: Some bacteria may contain storage bodies that can be stained. One example is the Gram positive bacilli Corynebacterium, which stores phosphate in structures called “volutin” or metachromatic granules that are housed within the cell membrane. Various staining methods are used to visualize intracytoplasmic bodies in bacteria, which often provide an identification clue when observed in cells.

Endospore Stain

Endospores are dormant forms of living bacteria and should not be confused with reproductive spores produced by fungi. These structures are produced by a few genera of Gram-positive bacteria, almost all bacilli, in response to adverse environmental conditions. Two common bacteria that produce endospores are Bacillus or Clostridum. Both live primarily in soil and as symbionts of plants and animals, and produce endospores to survive in an environment that change rapidly and often.

The process of endosporulation (the formation of endospores) involves several stages. After the bacterial cell replicates its DNA, layers of peptidoglycan and protein are produced to surround the genetic material. Once fully formed, the endospore is released from the cell and may sit dormant for days, weeks, or years. When more favorable environmental conditions prevail, endospores germinate and return to active duty as vegetative cells.

Mature endospores are highly resistant to environmental conditions such as heat and chemicals and this permits survival of the bacterial species for very long periods. Endospores formed millions of years ago have been successfully brought back to life, simply by providing them with water and food.

Because the endospore coat is highly resistant to staining, a special method was developed to make them easier to see with a brightfield microscope. This method, called the endosporestain, uses either heat or long exposure time to entice the endospores to take up the primary stain, usually a water soluble dye such as malachite green since endospores are permeable to water. Following a decolorization step which removes the dye from the vegetative cells in the smear, the counterstain safranin is applied to provide color and contrast. When stained by this method, the endospores are green, and the vegetative cells stain pink, as shown in Figure 7.

Figure 7. Bacterial cells with endospores, stained with the endospore stain.

Figure 8. Bacilli with endospores viewed by phase-contrast microscopy.

Although endospores themselves are resistant to the Gram stain technique, bacterial cells captured in the process of creating these structures can be stained. In this case, the endospores are seen as clear oval or spherical areas within the stained cell. Endospores can also be directly observed in cells by using phase contrast microscopy, as shown in Figure 8.

Because many differential staining methods require several steps and take a long time to complete, we will not be performing all of the differential staining methods discussed above.

Pre-stained slides will be used to visualize bacterial capsules, metachromatic granules, and acid-fast bacilli. Obtain one slide of each of the three bacteria listed in the table below. As you view these slides, make note of the “highlighted” structures. Your environmental isolate may have one or more of these cellular features, and learning to recognize them will aid in identification. These should all be viewed using the oil immersion objective lens.

BacteriumStainDescription or sketch of cells with the specified feature
Flavobacterium capsulatumCapsule stain
Corynebacterium diphtheriaeMethylene blue(metachromatic granules)
Mycobacterium tuberculosisAcid fast stain

Gram Stain

All staining procedures should be done over a sink. The Gram stain procedure will be demonstrated, and an overview is provided in Table 1.

Table 1. Gram stain procedural steps.
Primary stain(crystal violet)Add several drops of crystal violet to the smear and allow it to sit for 1 minute. Rinse the slide with water.Both Gram-positive and Gram-negative cells will be stained purple by the crystal violet dye.
Mordant (iodine)Add several drops of iodine to the smear and allow it to sit for 1 minute. Rinse the slide with water.Iodine “sets” the crystal violet, so both types of bacteria will remain purple.
Decolorization (ethanol)Add drops of ethanol one at a time until the runoff is clear. Rinse the slide with water.Gram-positive cells resist decolorization and remain purple. The dye is released from Gram-negative cells.
Counterstain(safranin)Add several drops of safranin to the smear and allow it to sit for one minute. Rinse the slide with water and blot dry.Gram-negative cells will be stained pink by the safranin. This dye has no effect on Gram-positive cells, which remain purple.

A volunteer from your lab bench should obtain cultures of the bacteria you will be using in this lab, as directed by your instructor. One of the cultures will be a Gram positive bacterium, and the other will be Gram negative. Below, write the names of the bacteria you will be using, along with the BSL for each culture:


Obtain two glass slides, and prepare a smear of each of the two bacterial cultures, one per slide, as demonstrated. Allow to COMPLETELY air dry and heat fix. Stain both smears using the Gram stain method. Observe the slides with a light microscope at 1,000X and record your observations in the table below.

Name of cultureGram stain reactionCellular morphologyArrangement

Gram Stain “Final Exam”: prepare a smear that contains a mixture of the Gram-positive AND Gram-negative bacteria by adding a small amount of each bacterium to a single drop of water on a slide. Heat fix the smear and Gram stain it. You should be able to determine the Gram stain reaction, cellular morphology and arrangement of BOTH bacteria in this mixed smear. Your instructor may ask to see this slide and offer constructive commentary.

HomeEndospore Stain

Only a few genera of bacteria produce endospores and nearly all of them are Gram-positive bacilli. Most notable are Bacillus and Clostridium species, which naturally live in soil and are common contaminants on surfaces. The growth of Clostridium spp. is typically limited to anaerobic environments; Bacillus spp. may grow aerobically and anaerobically. Endospore-forming bacteria are distinct from other groups of Gram positive bacilli and distinguishable by their endospores.

An overview of the endospore stain procedure is provided in Table 2.

Table 2. Endospore stain procedural steps.
Primary stain(malachite green)Add several drops of malachite green to the smear and allow it to sit for 10 minutes. If the stain starts to dry out, add additional drops.Vegetative cells will immediately take up the primary stain. Endospores are resistant to staining but eventually take up the dye.
Decolorization(water)Rinse the slide under a gentle stream of water for 10-15 seconds.Once the endospores are stained, they remain green. A thorough rinse with water will decolorize the vegetative cells.
Counterstain(safranin)Add several drops of safranin to the smear and allow it to sit for 1 minute. Rinse the slide and blot dry.Decolorized vegetative cells take up the counterstain and appear pink; endospores are light green.

After staining, endospores typically appear as light green oval or spherical structures, which may be seen either within or outside of the vegetative cells, which appear pink.

The shape and location of the endospores inside the bacterial cells, along with whether the sporangium is either distending (D) or not distending (ND) the sides of the cell, are important characteristics that aid in differentiating among species (see Figure 9).

Figure 9

  1. Oval, central, not distended (ND)
  2. Oval, terminal, ND (and parasporal crystal)
  3. Oval, terminal, distended (D)
  4. Oval, central, D
  5. Spherical, terminal, D
  6. Oval, lateral, D

Endospores are quite resistant to most staining procedures; however, in a routinely stained smear, they may be visible as “outlines” with clear space within. If you observe “outlines” or what appear to be “ghosts” of cells in a Gram stained smear of a Gram-positive bacilli, then the endospore stain should also be performed to confirm the presence or absence of endospores.

A volunteer from your lab bench should obtain bacterial cultures for endospore staining, as directed by your instructor. Note that these will all be species of Bacillus. Prepare smears and stain each using the endospore staining technique. Observe the slides and note the shape and location of the endospore and the appearance of the sporangium (swollen or not swollen) in the table below:

Name of cultureEndospore ShapeLocationSporangium

In addition, choose ONE of the cultures from above and Gram stain it. Record your results below in the spaces provided:

Name of Gram stained culture: __________________________________________________

Gram stain reaction and cellular morphology: ______________________________________

Are endospores visible in the Gram stained smear? _________________ If you see endospores, describe how they appear in the Gram stained preparation, and how this is similar to and different from what you see in the endospore stained preparation.

Lab Experiment #5 Differential Staining

Through the process of differential staining, there are distinct differences between the cell walls of gram-positive and gram-negative bacteria. In the case of gram-positive bacteria, the cell wall is comprised of 60-90% peptidoglycan and is very thick. There are numerous layers of teichoic acid bound with peptidoglycan thereby creating very thick cell membranes which causes the cell wall to take up large quantities of basic dye and appears purple. (Hands-on-Labs. (2012)). Conversely, gram-negative bacteria cell walls are much thinner with an outer cell membrane composed of phospholipids and only 10-20% peptidoglycan. Therefore, it appears pink via the process of differential staining. The difference in color can be attributed to the thinner cell wall and the decolorization process that occurs with the application of the mixture of ethyl alcohol and acetone.

Utilization of Grams mordant solution of iodine on the bacteria generates an insoluble complex, acting as a binding agent. Mordant is defined as a substance, typically an organic oxide that combines with a dye or stain and thereby fixes it in a material (Wikipedia. (2013)). When the mordant was applied, a decolorizer (acetone/alcohol) was applied to differentiate the thicker cell walled structures (those that remained purple) from the thinner cell walled structures (those that turned pink).

The differential staining method enabled visualization of the thicker cell walled, gram-positive bacteria Staphylococcus and Lactobacillus, as well as the purple staining of the yeast S. cerevisiae. The color of S. cerevisiae is noteworthy, as it is a fungus. The rules of differential staining do not apply. The fungus simply picks up the first color/stain utilized, in this case purple (McCarthy, Tom. (2014)). In the differential staining procedure, gram-negative E. coli was visualized and appeared pink.

In the case of gram-positive staining, the Lactobacillus gram positive rods were seen. This bacteria exists in many popular foods including yogurt, cheese, and the fermentation of beer and wine. Alternatively, the bacteria can have negative impact on the human body in the form of infections, commonly involving the urinary tract. Another gram-positive bacteria, Staphylococcus, exists in the nose and on the skin of humans. Found in these locals, it is generally benign, until there is a disruption or injury in the skin that introduces it systemically. (Stoppler, Melissa Conrad MD). Staphylococcus can be insidious and potentially life-threatening if left untreated, causing sepsis if left to run rampant. A yeast delineated as S. cerevisiae appeared purple in this experiment. This would lead one to believe that it is gram-positive as well. However, as a fungus, the rules do not apply, as its cellular structure is different and it takes up whatever color is first introduced. The only traceable source of infection linked to S. cerevisiae is via the use of S. saccromyces as a probiotic during the course of intensive antibiotic treatment and the subsequent proliferation of S. cerevisiae.

Finally, in the case of gram-negative bacteria, E. coli is visualized as pink capsular colonies in the differential staining slides. Utilized as a main fermenting agent with tremendous genetic diversity, it has historically been used in the making of beer, bread and rice wine. (US National Library of Medicine. May 2010)). While E. coli does exist in the human body, introduction of harmful variants through ingestion of contaminated food, for example, can lead to (bloody) diarrhea, dehydration and kidney failure.

Hands-on-Labs. (2012). A Laboratory Manual of Small-Scale Experiments for the Independent Study of Microbiology. Englewood, CO.

Lab 5 Cell Structure And Staining Using Microscopy 1

Instructions: Please download this MSWord document to your computer and answer the questions as asked. Then save the document and upload it to Bb using the Assignment feature provided. This assignment is worth a total of 100 points – there are 20 questions worth 5 points each.

In Lab 3 you were introduced to microscopy. In this lab you will be adding to that experience by reviewing the differences in cell structure for Prokaryotes and Eukaryotes (previously covered in Lecture Unit #1) and learning about staining of microbes.

Part I. Smear Preparation in Mastering Microbiology.
Log in to MM, go to the Study Area, and then to Microlab Tutor “Smear Preparation”. After viewing the short video, answer the following question: 1. List the major steps in smear preparation

If the slide is not clean, clean the slide.
Label the slide.
Make a dime size circle
Place a drop of saline solution on to the slide.
Sterile the loop/or use a sterile loop and obtain culture of the bacteria from the petri dish or tube. Harvest just enough culture. (only use a tiny amount of culture preparing a smear) Spread the bacteria on the glass slide. Set the slide to air dry. Either heat fix or methanol fix. (do not fix it both ways)

Stain the slides.
Properly dispose of any wastes.(waste methanol, materials contaminated with bacteria, broken glass)

Part II. Atlas Sections
In your Lab Atlas you will need to read Section 5: Bacterial Cellular Morphology and Simple Stains and Section 6: Bacterial Cell Structures and Differential Stains and then answer the following questions: 2. What is the third important feature of microscopy? Why?

Third important feature of microscopy is contrast. To be visible, the specimen must contrast with the background of the microscope field.

3. A simple stain will help determine these 3 features of the specimen on the slide: Cell morphology, size and arrangement then may be determined.

4-A. What part of the stain is responsible for its COLOR?

Chromophore is responsible for the stain’s color.

4-B. Name 3 examples of basic stains:
Methylene blue, crystal violet, and safranin.

5. List the three things that heat fixing a smear prior to staining achieves: Heat-fixing kills most of the bacteria, makes them adhere to the slide, and coagulate cytoplasmic membranes to make them more visible. It also distorts the cells to some extent.

6. What are the three types of cell morphology in bacteria?

Spheres (cocci, singular coccus)
Rods (bacilli, singular bacillus)
Spirals (spirilla, singular spirillum)

7. Cell arrangement is an important feature that aids in identification of bacteria. What is the difference between staphylococcus and streptococcus?

If the cell continue to divide in the same plane and remain attached, they exhibit a streptococcus arrangement. If the division planes of a coccus are irregular, a cluster of cells is produced to form a staphylococcus.

8. What is the purpose of the negative stain? What example is cited in your Atlas?

The negative staining technique is used to determine morphology and cellular arrangement in bacteria that are too delicate to withstand heat-fixing. A primary example is spirochete.

9. What is the purpose of the Gram stain? What part of the bacterial cells determines the outcome?

The purpose of the Gram Stain is to distinguish Gram-positive and Gram-negative cells. Different cell wall construction of Gram-negative and Gram-positive cells determine the ability to resist decolorization or not.

10. List the 4 steps of the Gram Stain naming the chemical used in each step:

a. Primary stain- crystal violet and.
b. Add iodine as a mordan ( form a crystal violet-iodine complex)
c. Decolorization – ( alcohol or acetone)
d. Counter-stain – Safranin

11. What is the purpose of these differential staining techniques:
a. Acid-fast It is used as a differential stain to detect cells capable of retaining a primary stain when treated with an acid alcohol. It is important differential stain used to identify bacteria in the genus Mycobacterium., coccidian parasites, acid-fast bacilli.

b. Capsule Stain It is a differential stain used to detect cells capable of producing an extracellular capsule. Capsule production increases virulence in some microbes such as the anthrax bacillus and the pneumococcus.

c. Endospore Stain The spore stain is a differential stain used to detect the presence and location of spores in bacterial cells. Only a few genera produces spores.

Part III. Staining in Mastering Microbiology.
Returning to Mastering Microbiology, go to the Study Area, Microlab Tutor and view the video on “Gram Staining”. Then go to “Microbiology Animations with Quizzes” and work your way through “Staining”. Then answer the following questions by circling the letter of the correct answer: 12. Which type of staining method would you use to determine endospore-forming cells from non-endospore-forming cells?

a. Specialized b. Differential c. Regular d. Simple

13. Which of the following is a characteristic of simple stains?
a. They stain specific structures of a bacterial cell.
b. They stain specific structures of a bacterial cell AND can be rinsed with water.
c. They can be rinsed with water.
d. They are a basic stain.
e. They are a basic stain AND can be rinsed with water.

Part IV. Structure and Microscopy – Lab 5 from eScience Lab Manual. Read the background information on p. 76-79. Following the instructions, prepare your Agar Plate and inoculate it with specimens from two different surfaces as directed. 14. Photograph of your plate after inoculation and incubation. You may insert your photograph here or you may upload it as separate attachment in Bb.

15. What 2 surfaces did you swab for your samples? Which sample provided the most growth?

I wanted to take this opportunity and try different surfaces. I have swabbed my trashcan and my cellphone. I was expecting trashcan to have higher bacteria. Both of the surfaces I swabbed did not end up the amount of growth I expected. I have observed 3 different types of colonies growing on the trashcan though. (My incubation time was 6 days)

Part V. To simulate the Gram Stain process, go the website listed on Bb for the “Virtual Gram Stain.” To run the process, click on “View Example” and then start by clicking on the 1st Test Tube to be used in the Gram Stain process. For each subsequent step, click on the next tube to be used in the process.

I have completed this fun little practice. It helped to clarify the staining procedure.

Part VI. Returning to the eScience Lab Manual, prepare your three slides as directed on p. 81-83. In the absence of a microscope with which to view your slides, refer to the color photographs of bacteria in Section 5 and 6 in your Lab Atlas.

16. Photograph your slides and insert the clearly labeled photographs or upload them as separate attachments on Bb.

17. In the Gram Stain, what different characteristic(s) exist between the two groups that allow for the different staining conditions?

The cell envelopes of most bacteria fall into one of two major groups. Gram-negative bacteria are surrounded by a thin peptidoglycan cell wall, which itself is surrounded by an outer membrane containing lipopolysaccharide. Gram-positive bacteria lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the Gram-negatives.

18. Why was the Gram iodine added to the Gram staining technique?

Gram iodine is added to the Gram staining as a mordant to enhance crystal violet staining.

19. Why is a counterstain (safranin) added to the Gram staining procedure?

After decolorization procedure, Gram-positive cells remain purple whereas Gram-negative cells colorized by the counterstain Safranin. Upon successful completion of a Gram stain, Gram-positive cells appear purple and Gram –negative cells appear reddish-pink.

20. What are the advantages of performing a negative stain versus a simple stain for visualizing bacteria?

Even too delicate bacteria can be treated with negative stain. Also when the accurate size is crucial, a negative stain can be used because it produces minimal cell shrinkage. It provides a more detailed assessment of a microbe’s morphology than simple staining does because beckground rather than the microbe is stained. The microbe’s ultrastructure is preserved and it outlines the cells and makes it highly visible.

Associate Engineer - Civil (PE Required - 2.5% differential applies)

NOTE Regarding Salary: A 4% COLA (Cost of Living Adjustment) will apply as of 6-28-21
Pacheco Project Delivery Unit (Position Code 0588)
Overview:Valley Water's Pacheco Reservoir Expansion Project (PREP) continues to develop through planning, environmental impact analysis, engineering design, and permitting with construction anticipated to begin in late 2024. The PREP includes expanding the existing Pacheco Reservoir with new construction of a dam upstream of the existing dam, a spillway structure, inlet/outlet works, pipelines, a pump station, roads, power supply, and associated improvements. This Associate Engineer (Civil) position is an important piece of the PREP team in charge of providing technical oversight and review of deliverables such as plans, specifications, and technical memorandums organizing field investigations overseeing consultant work activities. coordinating with other agencies/organizations and communicating across technical groups with Valley Water staff and consultants. Engineering support during construction as a PE subject matter expert will including the following tasks: complete submittal reviews respond to requests for information and interpret project drawings, specifications, and contract documents.

Key Responsibilities include, but are not limited to:

  • Review engineering work by consultants and/or Valley Water staff, provide technical review, guidance, and direction.
  • Lead consultants and/or Valley Water staff in the preparation of planning documents, design drawings, technical deliverables, and the management of construction projects.
  • Coordinate and communicate with external agencies to develop technical project elements.
  • Represent Valley Water in public meetings, present recommendations to the Board, and other inter-agency coordination.

Ideal Candidate's Background Includes:
Applicants whose experience and background best match the ideal experience, knowledge, skills, abilities, and education are considered ideal candidates for the position. To determine the top candidates, each applicant will be assessed based on the ideal candidate criteria listed below.

Ideal Experience:
Minimum four (4) years of professional engineering experience.

Ideal Skills and Abilities:

  • Plan, organize, schedule, assign, train, review, and evaluate the work of staff.
  • Recommend and implement work plans and effectively manage engineering projects and project teams.
  • Apply engineering principles, practices, concepts, and standards to engineering problems.
  • Apply water resources principles, practices, concepts, and standards to water supply planning, operations, and management, groundwater monitoring, contamination, and protection, and water quality problems.

Ideal Knowledge:

  • Principles, practices, concepts, and standards of civil engineering as applied to assigned field/specialty of engineering, water resources management, natural resource management and planning, engineering, hydrogeology, geology, hydrology, hydraulics, and/or environmental sciences.
  • Principles and practices of project budgeting, cost estimation, funding, project management, and contract administration.
  • Principles and practices of numerical modeling and analysis, forecasting and risk analysis, and statistical analysis.
  • Practices of researching engineering and design issues, evaluating alternatives, making sound recommendations, and preparing and presenting effective and technical reports.
  • English usage, grammar, spelling, vocabulary, and punctuation.
  • Principles and techniques for providing customer service by effectively dealing with the public, vendors, contractors, and Valley Water staff.

Ideal Training and Education:
Graduation from an accredited four-year college or university with major coursework in civil engineering or a field related to assigned functional area(s).
Possession of a valid California Engineer-in-Training (EIT) Certificate with two (2) years of associated paraprofessional engineering experience.

Required License or Certificate

  • Possess and maintain a Professional Engineer (PE) License issued by the California Board of Professional Engineers, Land Surveyors, and Geologists.
  • Possession of, or ability to obtain, an appropriate, valid California driver's license. Individuals who do not meet the driver's license requirement due to a disability will be considered for a reasonable accommodation on a case-by-case basis.

To review the Classification Specification, please click here (Download PDF reader)

Selection Process

(1) The selection process may include one or more of the following: application review, application assessment, performance exercise, written exercise and/or interview.
(2) The Employment Application, Qualifying Information Questions and/or Supplemental Questions will be evaluated based on the ideal candidate criteria listed above. Resumes are highly recommended.

NOTE: Position and start date is subject to availability of funds. Valley Water retains the right to repost this position as deemed necessary.
Consideration may be given to existing applicant pools within the same classification.

Valley Water's Equal Opportunity Non-Discrimination Policy is available for review upon request.

Valley Water will make reasonable efforts in the examination process to accommodate persons with disabilities. Please advise Human Resources in advance of any special needs by calling 408-630-2260.

Please be aware that once submitted all application materials become the property of Valley Water and will not be returned. Human Resources staff are not authorized to make copies of application materials for applicants.

General Concepts, Prostate

Α-methylacyl-CoA-racemase (AMACR, P504S)

Overexpressed in prostate cancer with marked differential staining between malignant (positive) and benign (negative or weak expression) glands ○

Identified in gene expression array studies

Highly sensitive, reportedly positivity in ∼ 75-95% of carcinomas

Similar immunoreactivity pattern seen in HGPIN (reported 56-100%)

Negative or weakly expressed in some prostatic carcinomas: 5-25% typical, 30% atrophic, 32-38% foamy gland, 23-30% pseudohyperplastic, and up to 29% hormone-treated carcinomas

Expressed in benign lesions: 35-58% nephrogenic adenoma, up to 29% luminal staining in partial atrophy and postatrophic hyperplasia, 2-36% benign glands, 18% atypical adenomatous hyperplasia

Not specific for prostatic origin in metastatic setting –

Expressed by variety of other malignancies

Contributions of City-Specific Fine Particulate Matter (PM 2.5) to Differential In Vitro Oxidative Stress and Toxicity Implications between Beijing and Guangzhou of China

Growing literature has documented varying toxic potencies of source- or site-specific fine particulate matter (PM2.5), as opposed to the practice that treats particle toxicities as independent of composition given the incomplete understanding of the toxicity of the constituents. Quantifying component-specific contribution is the key to unlocking the geographical disparities of particle toxicity from a mixture perspective. In this study, we performed integrated mixture-toxicity experiments and modeling to quantify the contribution of metals and polycyclic aromatic hydrocarbons (PAHs), two default culprit component groups of PM2.5 toxicity, to in vitro oxidative stress caused by wintertime PM2.5 from Beijing and Guangzhou, two megacities in China. PM2.5 from Beijing exhibited greater toxic potencies at equal mass concentrations. The targeted chemical analysis revealed higher burden of metals and PAHs per unit mass of PM2.5 in Beijing. These chemicals together explained 38 and 24% on average of PM2.5-induced reactive oxygen species in Beijing and Guangzhou, respectively, while >60% of the effects remained to be resolved in terms of contributing chemicals. PAHs contributed approximately twice the share of the PM2.5 mixture effects as metals. Fe, Cu, and Mn were the dominant metals, constituting >80% of the metal-shared proportion of the PM2.5 effects. Dibenzo[ a, l]pyrene alone explained >65% of the PAH-shared proportion of the PM2.5 toxicity effects. The significant contribution from coal combustion and vehicular emissions in Beijing suggested the major source disparities of toxicologically active PAHs between the two cities. Our study provided novel quantitative insights into the role of varying toxic component profiles in shaping the differential toxic potencies of city-specific PM2.5 pollution.


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2. Experimental procedures

2.1. Chemicals and reagents

All reagents used in cell culture were cell culture-tested. The Dulbecco's modified Eagle medium (DMEM) with Glutamax™ and low glucose (1 g/L), fetal bovine serum (FBS), trypsin 0.05% solution in phosphate buffered saline (PBS), Fungizone ® Antimycotic (amphotericin B), penicillin-streptomycin solution (Pen-Strep) solution, and Dulbecco′s phosphate buffered saline (DPBS) (10×) were obtained from Invitrogen. The hydrogen peroxide (H2O2) used in oxidative stress stimulation was obtained from Sigma-Aldrich.

The reagents used in the differential alkylation procedure have different origins: the iodoacetamide was obtained from Sigma-Aldrich, the 40% acrylamide/bis-acrylamide solution (37.5:1) from BioRad and the dithiothreitol (DTT) from GE Healthcare Life Sciences. For in gel digestion, the precast polyacrylamide gels 𠇄�% TGX Stain-Free Gel” and all the buffers, including the Laemmli buffer, used in the electrophoresis were obtained from Bio-Rad, and the Trypsin Modified Sequencing Grade used in protein digestion, was obtained from Roche Diagnostics.

The reagents used in MS analysis were all high-quality chemical or reagents (ACS Reagent Chemicals & Lab Grades). Formic acid (FA) was obtained from AMRESCO and water, methanol and acetonitrile (ACN) from Fisher. Ortho-phosphoric acid and ammonium sulfate were from MERK and ammonium bicarbonate from Fluka.

2.2. Experimental design and statistical rationale

Two types of experiments were performed in this study: i) an initial validation of the method by an in-vitro assay using a redox-regulated recombinant protein subjected to direct oxidation with H2O2 ii) application of the method in a biological context by assessing the oxidative status of the proteins secreted under control and oxidative stress conditions. For the in-vitro study, two recombinant forms of the protein DJ-1 were used: the [WT]DJ-1 and [C106DD]DJ-1, for the wild-type (non-oxidized) and a constitutive C106 oxidized form of the protein, respectively. Both recombinant proteins were analyzed under reduced condition (control condition) and after a direct oxidation by H2O2 (positive control for cysteine oxidation). Each in-vitro reaction was performed in a total of four replicates (each reaction was performed with a different aliquots of the recombinant protein). Cells’ secretomes were collected from HeLa cells under control and oxidative stress conditions (induced by stimulation with H2O2) in a total of four biological replicates each. Each secretome (also known as conditioned medium) was split into three parts to be subjected to the three reactions of alkylation (see subheading 𠇍ifferential alkylation” for details). A recombinant protein (MBP-GFP) was added to the media after collection, to be used as internal standard [20].

Different statistical approaches were applied depending on the data being analyzed. The comparison tests applied were mainly parametric methods (Student's t-test and ANOVA) with statistically significant differences being considered for p-values below 0.05. No multiple comparisons correction was applied. Data normality was assessed by different methods, including Q-Q plots and Shapiro-Wilk test, and homogeneity of variances was evaluated by Levene's test. Outliers’ detection was performed by the ROUT method.

Both experimental design and the respective data analysis will be detailed in the following subsections.

2.3. Recombinant DJ-1 production

2.3.1. DNA constructs

The synthetic DNA coding for the human protein DJ-1 with the codons optimized for E. coli expression was chemically synthetized from GeneArt ® Gene Synthesis (Invitrogen) and amplified by PCR to include the restriction sites for NheI and XhoI at 5′- and 3′-ends, respectively using the forward primer 5’-GCTAGCAAACGTGCACTGGTTATTCTG-3’ and the reverse primer 5’-CTCGAGTCAATCTTTCAGAACCAGCGGTG-3’. The purified product was cloned into pGEM ® -T-Easy plasmid (Promega) and subcloned to pSKB-3 expression vector (DJ-1_pSKB-3) after digestion of both with NheI and XhoI (New England Biolabs, Inc.), in frame with a N-terminal hexahistidine-tag and a TEV (tobaco etch virus) recognition site, for the expression of a recombinant DJ-1 with a TEV-cleavable His-tag. The constitutively C106-oxidized mutant of DJ-1 was generated by replacing the cysteine 106 residue by two aspartic acid residues (C106DD) using the Quick-Change Site-Directed Mutagenesis kit (Stratagene) and the primers 5′-CGCAAAGGTCTGATTGCAGCAATT GATGAT GCAGGTCCGACCGCACTGC-3′ (forward) and 5′-GCAGTGCGGTCGGACCTGC ATCATC AATTGCTGCAATCAGACCTTTGCG-3′ (reverse) (mutation underlined). All positive clones were selected by restriction analysis and confirmed by DNA sequencing.

2.3.2. Expression and purification

The DJ-1_pSKB-3 constructions (for both WT and C106DD mutant) were transformed into competent E. coli BL21star (DE3) strain and transformed cells were allowed to grow at 37 ଌ in LB supplemented with 50 μg/mL kanamycin until an optical density at 600 nm of 0.5 was reached, after which the protein expression was induced for 16 h by the addition of IPTG to a final concentration of 1 mM. After protein expression, the pellet of cells was resuspended in 20 mM sodium phosphate, 500 mM NaCl, 10 mM Imidazole, pH 7.2 and disrupted. The cellular extract was clarified by centrifugation, the supernatant was filtered through 0.2 µm syringe filters and the protein was applied to a 5 mL HisTrap HP column (GE Healthcare) pre-equilibrated in the same buffer. Protein elution was obtained by stepwise increasing of imidazole concentration up to 500 mM (50, 100, 300 and 500 mM) and the fraction containing the higher amount of the protein of interest was further purified by size-exclusion chromatography with a HiLoad 26/600 Superdex 200 prep grade column (GE Healthcare Life Sciences) and eluted using PBS (8 mM K2HPO4, 2 mM NaH2PO4·H2O, 150 mM NaCl).

2.4. Conditioned medium collection

HeLa cells were seeded at 12 ×� 3 cell/cm 2 in 148𠋼m 2 plates (Corning) in a total of 4 plates per replicate. After 48 h in culture (37 ଌ with 5% of CO2/95% air and 95% humidity) the culture medium (DMEM medium with 10% FBS) was discarded and cells were washed twice with warm PBS to remove the remaining FBS. Then, the culture medium was changed to DMEM without FBS (control condition) or to a solution of 1 mM of H2O2 in DMEM without FBS (stress condition) for 40 min, after which the medium was changed again to DMEM without FBS. The conditioned media were collected 24 h after the stimulation. The collected media were centrifuged at 290g, for 5 min at 4 ଌ to remove cell debris, and then split into three parts for differential alkylation. A recombinant protein (MBP-GFP) was added to the media after collection, to be used as internal standard in the quantitative analysis [21]. Each condition was performed in a total of four replicates.

2.5. Differential alkylation

2.5.1. In-vitro assay with the recombinant DJ-1 proteins

Ten micrograms of each recombinant protein ([WT]DJ-1 and [C106DD]DJ-1) were used per reaction (Supplementary Fig. S1-A). The first alkylation steps were performed using iodoacetamide (IAM) at a final concentration of 66 mM (R1 and R2) and acrylamide (Acry) at 6% (v/v) (R3), and their concentrations were triplicated in the second steps (R3 in the case of IAM, and R1 and R2 for Acry) to compensate for the excess of DTT. DTT was added to a final concentration of 11 mM and the reduction and alkylation steps were performed using ultrasonication for 10 min. Ultrasonication was performed in a 750 W Ultrasonic processor with cuphorn using 20% intensity and pulses ON/OFF of 1 s each. The reagents from the first alkylation were removed using cut-off filters of 5 kDa (Vivaspin500, Sartorius) followed by a washing step with 0.5 M TEAB, and this step was repeated before the protein digestion to remove the excess of reducing and alkylating reagents. To promote the in-vitro oxidation of the recombinant proteins prior to the differential alkylation, they were incubated with 1 mM of hydrogen peroxide for 30 min at room temperature. Each reaction was performed in a total of four replicates and all samples were subjected to liquid digestion. In addition, more reactions were performed using a single alkylating reagent, with or without reduction of the samples prior to alkylation. These reactions were only used for peptide identification.

2.5.2. Conditioned media

The conditioned medium of each sample was split into three parts, one of them to be immediately subjected to the alkylation step using iodoacetamide (to block the reduced cysteines, Fig. 1 A – R1) while the other two parts were subjected to reduction with DTT prior to the alkylation step (R2 and R3). All the reactions, except the alkylation with iodoacetamide in R1, were performed after the concentration of the media to 600 µL using the cut-off filters of 5 kDa (Vivaspin20, Sartorius). Prior to media concentration, acetonitrile was added to all samples to a final concentration of 20% (v/v) [2] to promote some protein denaturation, and iodoacetamide was added to the R1 part at a final concentration of 66 mM. After the concentration of the medium, cysteines were reduced by the addition of DTT to a final concentration of 11 mM and alkylated with 66 mM of IAM (R2), 6% (v/v) of Acrylamide in R3 and 12% (v/v) of acrylamide in R1. All the reactions were performed in an ultrasonic bath (USC 1200 THD & THD/HF) at maximum power for 45 min.

Differential alkylation combined with SWATH-MS acquisition: oxSWATH. A) Schematic representation of the experimental workflow used to evaluate the proposed label free differential alkylation method. In this method, the differential alkylation (R1) is performed by using two widely used alkylating agents, iodoacetamide (IAM) and acrylamide (Acry), to block the reduced and the reversible oxidized cysteines, respectively. This reaction is compared with two additional reactions where sample is completely reduced prior to alkylation (R2 and R3 with IAM and acrylamide, respectively) to access the total level of the cysteine peptides. Both cysteine containing peptides and peptides without cysteines are monitoring by SWATH-MS, to measure the changes in cysteine-redox state and total protein levels, respectively. B) Iodoacetamide reaction with free thiols. C) Acrylamide reaction with free thiols. D) Detection of pairs of cysteine-alkylated peptides acquired in the different and in the same SWATH-MS window. The selectivity of the method is verified through the analysis of representative LC-MS/MS chromatograms of two pairs of DJ-1 peptides DVVIC [IAA] PDASLEDAKK/DVVIC [Acryl] PDASLEDAKK (acquired in different windows – see Supplementary Table S2) and DVVIC [IAA] PDASLEDAK/DVVIC [Acryl] PDASLEDAK (acquired in the same window) in the R2 (samples only alkylated with IAA) and R3 (samples only alkylated with Acryl). For each peak group is presented the FDR value obtained using SWATH™ processing plug-in for PeakView. Retention time (TR) was adjusted for each sample and the XICs were obtained in a window of 4 min centered at the aligned retention time. See Supplementary Fig. S4 for detailed information regarding the areas integrated in the pair of peptides acquired in the same window.

After the differential alkylation, the concentrated conditioned media were precipitated using Trichloroacetic acid (TCA) – Acetone. Briefly, TCA was added to each sample to a final concentration of 20% (v/v), followed by an incubation at −� ଌ and centrifugation at 20,000g for 20 min. Protein pellets were washed and solubilized with ice-cold (� ଌ) acetone, followed by a centrifugation at 20,000g for 20 min [22]. The washed pellets were re-suspended in 2× Laemmli buffer, aided by ultrasonication and denaturation at 95 ଌ for 5 min [23].

2.6. Protein digestion

Recombinant DJ-1 samples were subjected to liquid digestion as described in Anjo et al. [24], without the alkylation step. The digestions were performed with trypsin in a 1:50 ratio, overnight at 37 ଌ.

The secretomes were subjected to in gel digestion using the short GeLC approach [24]. Seventy microliters of each sample were used for SWATH-MS analysis, and 20 µL of each replicate were combined to create representative pools of each group of samples to be used for protein identification. A total of 6 pools (two experimental conditions from 3 different alkylation reactions) were made and subjected to the short-GeLC approach. The peptides of each digested pooled sample were separated by high pH Reverse Phase Chromatography as presented in Silva et al. [21] and the 28 collected fractions throughout the chromatographic run were joined into 6 samples per pool, in order to increase the number of proteins and peptides identified (in particular cysteine containing peptides) [25].

2.7. Mass spectrometry analysis by SWATH mode

2.7.1. Acquisition

Samples were analyzed on an AB Sciex ® 5600 TripleTOF in two modes: information-dependent acquisition (IDA) for protein identification and library generation, and SWATH acquisition for quantitative analysis. Peptide separation was performed using liquid chromatography (nanoLC Ultra 2D, Eksigent ® ) on a MicroLC ChromXP™ C18CL reverse phase column (300 µm × 15𠂜m, 3 µm, 120 Å, Eksigent ® ) at 5 µL/min with a multistep gradient: 0𠄲 min linear gradient from 5% to 10%, 2� min linear gradient from 10% to 30%, and 45� min to 35% of acetonitrile in 0.1% FA. Peptides were eluted into the mass spectrometer using an electrospray ionization source (DuoSpray™ Source, AB Sciex ® ) with a 50 µm internal diameter (ID) stainless steel emitter (AB Sciex ® ).

Information dependent acquisition (IDA) experiments were performed in the single alkylated samples in the case of the recombinant DJ-1, and for each fraction of the representative pooled samples of the conditioned media. The mass spectrometer was set to scanning full spectra (350� m/z) for 250 ms, followed by up to 100 MS/MS scans (100� m/z from a dynamic accumulation time – minimum 30 ms for precursor above the intensity threshold of 1000 – in order to maintain a cycle time of 3.3 s). Candidate ions with a charge state between +𠂒 and +𠂕 and counts above a minimum threshold of 10 counts per second were isolated for fragmentation and one MS/MS spectra was collected before adding those ions to the exclusion list for 25 s (mass spectrometer operated by Analyst ® TF 1.7, AB Sciex ® ). Rolling collision was used with a collision energy spread of 5.

For SWATH-MS based experiments, the mass spectrometer was operated in a looped product ion mode with the same chromatographic conditions used as in the IDA run described above. The SWATH-MS setup was designed specifically for the samples to be analyzed (Supplementary Tables S1 and S2 for recombinant DJ-1 and conditioned media experiments, respectively). A set of 45 or 60 windows of variable width (for recombinant DJ-1 or conditioned medium, respectively) was constructed covering the precursor mass range of 350� m/z. A 200 ms survey scan (350� m/z) was acquired at the beginning of each cycle for instrument calibration and SWATH MS/MS spectra were collected from 100 to 1500 m/z for 70 or 50 ms (for recombinant DJ-1 or conditioned medium, respectively) resulting in a cycle time of 3.25 s, which is compatible with the acquisition of at least 8 points per chromatographic peak. The collision energy for each window was determined according to the calculation for a charge +2 ion centered upon the window with a collision energy spread of 15.

2.7.2. Protein identification and library generation

Protein identification was performed in ProteinPilot™ software (v5.0, AB Sciex ® ) using the Paragon™ Algorithm (, 4767, AB Sciex ® ). Recombinant DJ-1 proteins were searched against the complete database from SwissProt (released at February 2015, composed by 547,351 entries) and conditioned medium was searched against a database composed by the Homo sapiens database from SwissProt (released at February 2015, composed by 20,200 entries) and the sequences of the recombinant proteins used as internal standard (IS). The searches were performed with the following parameters: trypsin digestion and iodoacetamide or acrylamide as cysteine alkylating reagent, for R2 and R3 samples, respectively and a special focus option for gel-based approaches was used in the analysis of the conditioned medium. In the conditioned media experiments, the files acquired from control and stress conditions were combined into a single search per reaction of alkylation (R2 or R3). Mass tolerances and exceptions to cleavage rules like missed or semi-specific cleavages were defined automatically according with predefined probabilities. Briefly, probabilities of 0.75 and 0.00001 were set for missed cleavage and non-specific cleavages, respectively, and the mass tolerances were set in 0.05 ±𠂐.0011� and 0.1 ±𠂐.01� for precursor and fragment ions, respectively. An independent False Discovery Rate (FDR) analysis, using the target-decoy approach provided by ProteinPilot™, was used to assess the quality of the identifications. Positive identifications were considered when identified proteins and peptides reached a 5% local FDR.

A specific library of precursor masses and fragment ions was obtained from each identification file using the SWATH™ processing plug-in for PeakView™ (v2.0.01, AB Sciex ® ). The libraries were exported as text files to be manually adapted to the redoxomics analysis. For each library, the peptides containing alkylated cysteines were isolated and further combined with the peptides from the internal standard, this step generates cysteine specific libraries (one for the iodoacetamide alkylated peptides and one for acrylamide alkylated peptides). In addition, another library is created using only peptides without cysteines, to be used to determine the total levels of the proteins. In total, three libraries are used per redoxomics assay.

2.7.3. SWATH data file processing

Peptides were selected automatically from the library using the criteria described in Anjo et al. [24]. Up to 15 peptides were chosen per protein for the determination of the protein total levels, and all the peptides containing cysteines were used in the case of the cysteine specific libraries. SWATH quantitation was attempted for all proteins in the library files that were identified below 5% local FDR from ProteinPilot™ search. For each peptides, up to 5 target fragment ions (corresponding to a peak group) were automatically selected and scored [24]. Peak group confidence threshold was determined based on a FDR analysis using the target-decoy approach and the peptide that met the 1% FDR threshold in all the replicates (in the case of IS), or at least three replicates were retained for posterior analysis. The peak areas of the target fragment ions of those peptides were extracted across the experiment using a 4 min extracted-ion chromatogram (XIC) window adjusted in order to accommodate the entire chromatographic peaks. The retention time was adjusted to each sample using the IS peptides.

Total protein levels were estimated by summing all the transitions from all the peptides [24] without cysteine of a given protein, and for each protein the levels of its cysteine alkylated peptides were determined by summing all the transitions of that group of peptides. Peptides alkylated with iodoacetamide and acrylamide were analyzed in separate.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [26] via the PRIDE [27] partner repository with the dataset identifier PXD006802 and PXD006803 (for conditioned media and recombinant DJ-1 data, respectively, and it is also available as a public resource in the SWATHAtlas database ( with the deposit no. PASS01210.

2.8. Western blot

Conditioned media were spiked with the same amount of IS solution prior to culture medium concentration. The samples were denatured by boiling at 95 ଌ for 5 min, and the entire volume was loaded per lane and separated on 12.5% SDS-polyacrylamide gels using a mini-PROTEAN ® Tetra Electrophoresis System (Bio-Rad). Proteins were transferred to low fluorescence polyvinylidene fluoride (PVDF) membranes (TBT RTA TRANSFER KIT, Bio-Rad) using a Trans-Blot Turbo Transfer System (BioRad) during 30 min at a constant voltage of 25 V (with the current limited to 1𠂚). Following transfer, the membranes were blocked for 1 h at room temperature (RT) with 5% (w/v) skimmed milk powder in PBS-Tween 20 (PBS-T) [0.1% (v/v)]. The membrane was incubated sequentially with anti-DJ-1 (1:1000 ADI-KAM-SA100-E, Enzo Life Sciences, Inc.) and anti-GFP (1:1000 SICGEN – Research and Development in Biotechnology Ltd.) overnight at 4 ଌ followed by 1 h at RT, prepared in the blocking solution. Primary antibodies were removed, and membranes were extensively washed with PBS-T (3 times, 15 min under agitation each time). Blots were then incubated for 1 h at RT with the respective secondary antibodies conjugated with alkaline phosphatase (anti-mouse and anti-goat for DJ-1 and GFP, respectively) in 5% (w/v) skimmed milk powder dissolved in PBS-T followed by extensive washes as above. The membrane was firstly incubated with the DJ-1 specific antibody followed by re-probing of the membrane with antibody against a GFP specific antibody.

Protein-immunoreactive bands were developed using the 𠇎nhanced Chemifluorescence (ECF) detection system” (GE Healthcare) and visualized in a Molecular Imager FX System (Bio-Rad). The adjusted volumes (total intensities in a given area with local background subtraction) for each band were obtained using the Image Lab software (version 5.1, Bio-Rad). DJ-1 levels were normalized for GFP levels (used as internal standard) and the differences in the secretion of DJ-1 were evaluated by a Student's t-test performed in GraphPad Prism (version 6.01).

2.9. Data analysis

2.9.1. Differential proteomics analysis of the secretome

Differential secretome analysis was performed using the results from the total levels of the proteins. Protein total levels were normalized for the values of the internal standard, and the normalized values of the three reactions (R1, R2 and R3) were combined into a single value per replicate. The proteins altered between the two conditions were identified by an ANOVA test performed in InfernoRDN (version 1.1.5581.33355) using the log10 transformed values, and statistically significant differences were considered for p-values <𠂐.05. Data normality was assessed by the analysis of Q–Q plots [28] obtained in InfernoRDN [29].

2.9.2. Determination of the reduced and reversible oxidized fraction of the proteins

The iodoacetamide alkylated peptides (from R1 and R2) are used to determine the reduced fraction of a give protein, and the acrylamide alkylated peptides (R1 and R3) are used to determine the reversible oxidized levels of the proteins (Supplementary Fig. S2). The levels of the cysteine alkylated peptides are firstly normalized to the total levels of the correspondent protein within the same reaction of alkylation, i.e., the values calculated from the R1 are compared with the total protein levels in R1, and so forth. The normalized values are used to determine the relative proportion of the reduced fraction by calculating the ratio between the IAM-alkylated peptides in R1 and the IAM-alkylated peptides in R2 (correspondent to the total amount of free available cysteines). To determine the levels of the reversible oxidized fraction, a similar ratio is performed but using the Acry-alkylated peptides from R1 and the Acry-alkylated peptides from R3. The combination of these two values should be closer to one, therefore values smaller than one may be an indication of irreversible oxidations. To determine the total levels of each oxoform (reduced and reversible oxidized fraction expressed in procedure defined unit, p.d.u), the relative proportions obtained in each replicate are multiplied by the correspondent protein total levels (summed peptides’ intensity) in R1 reaction (the reaction common to the two types of alkylating agents). See Supplementary Fig. S2 for a schematic representation of these calculations.

2.9.3. Comparative analysis of the oxidative state of DJ-1

The reduced and oxidized fractions were compared between the experimental conditions ([WT]DJ-1 vs [C106DD]DJ-1 and Control vs Stress conditions) using student's t-tests with statistical significance considered for p-values below 0.05. Parametric assumptions (data normality and homogeneity of variance) were tested using Shapiro-Wilk Test and Levene's Test, respectively. All the tests were performed in IBM® SPSS® Statistics version 22. Outliers’ detection was performed in GraphPad Prism (version 6.01) using the ROUT method.

2.9.4. Differential redoxomics analysis

Comparative redoxomics analysis of the secretome was performed using the results from the reduced and reversible oxidized fractions, for both the relative and the total proportions. In the case of the total levels, data was further normalized using the internal standard to accommodate potential sample processing errors. The proteins altered between the two experimental conditions (control vs stress) were identified as indicated above for the differential proteome analysis of the secretome. The analysis was repeated for each type of results related to the oxidative state of the secreted proteins.

2.9.5. Functional analysis

Protein domain enrichment analysis was performed with FunRich (version 3.1.3) using Protein domain database, and statistically analyzed with hypergeometric test using FunRich human genome database as the background [30]. A Bonferroni corrected p-value <𠂐.05 indicates the sub-groups of protein domains that are significantly enriched in the sample against the background database (9952 entries). Reactome (version 57) [31] (available at was used to perform pathway enrichment analysis of the total list of altered proteins (considering their secretion levels or alteration in redox state) in the present study. Pathways were considered enriched for FDR analysis below 5%.

2.9.6. Row-clustered heat maps

Heat maps were done using GPRroX (version 1.1.15) using the standardized levels of the quantified proteins within all the replicates of the two reactions.


Tissue samples

We analyzed a cohort of 36 snap frozen colon samples, obtained from the Tumor Bank at the Instituto de Oncolog໚ Asturias (IUOPA, Asturias, Spain). 18 of the samples were from LSCRCs, while the remaining 18 were used as control and were from the corresponding surrounding healthy tissue from the same patients. Diagnoses were carried out according to the World Health Organization (WHO) criteria using hematoxylin-eosin-stained slides and the snap frozen tissues were stored at −� ଌ prior to isolation of the RNA. Informed written consent of all the patients was obtained, and the study was approved by the Ethics Committee on Clinical Investigation of the Hospital Universitario Central de Asturias.

Total RNA isolation and cDNA synthesis

Tissue fragments (20� mg) were homogenized using a polytron PT 2100 (Kinematica Inc. Bohemia, NY), and RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany), and processed as previously described [21]. cDNA synthesis was carried out using the High Capacity cDNA Transcription Kit (Applied Biosystems, Foster City, CA, USA). The reactions were performed and the products cleaned and stored as previously described [21].

Quantitative real-time polymerase chain reactions (qRT-PCR)

qRT-PCR reactions, and analysis of amplimer products were carried out accordingly to methods already detailed [21]. Actin was used as the control gene to normalize individual gene expression.

Data analysis

Statistical analysis of the data and expression of the values of differential transcription were performed as previously described [21].

The overall survival (OS) and cumulative probability analyses were performed using the Kaplan–Meier method and the survival curves were compared by the log Rank test, using IBM® SPSS® Statistics V.21.

Tissue microarray construction and immunohistochemistry

Representative tumor regions were identified in each sample and selected to make a tissue microarray containing three tissue cores from each sample of LSCRC. After 5 min at 60 ଌ the tissue microarray blocks were cut in 4 μm thick sections, ready for immunohistochemical techniques. Tissue sections were treated, prepared and immunostained as previously described [21]. For the detection of chondroitin 6-sulfotransferase-2, syndecan 1, CD117, NDST1 and glypican-4, sections were heated in high pH Envision FLEX target retrieval solution at 65 ଌ for 20 min and then incubated for 20 min at room temperature in the same solution. To detect perlecan, CS, HS2ST1, UST, CS, CS4ST, the same procedure was followed except that the final step was omitted and the sections were instead incubated overnight at 4 ଌ in a humid chamber with primary antibodies. The antibodies and the dilution are detailed in the Additionalਏileਁ.

After the first incubation, sections were rinsed in the same buffer, and incubated with the following secondary antibodies anti-rabbit, anti-mouse EnVision system-labelled polymer (DakoCytomation) and anti-goat diluted 1:100 (Santa Cruz Biotechnology) for 1 h at room temperature. Finally, the sections were washed and the immunoreaction visualized using 3𠄳� as a chromogen. The sections were studied and photographed under a light microscope (Eclipse 80i Nikon Corporation, Tokyo, Japan).

Isolation of Bacteria in Pure Culture | Microbiology

The below mentioned article provides a short-note on the isolation of bacteria in pure culture. The methods used to isolate the bacteria in pure culture are: 1. Streaking or Plating 2. Dilution and Plating 3. Use of Selective Medium 4. Differential Sterilisation by Chemicals 5. Differential Sterilisation by Heat and 6. Inoculation of a Susceptible Animal.

To determine accurately the specific causative agent of the disease by its diagnostic characters, the microbiologist must isolate a single bacterium, in pure form, from other bacteria with which it is mixed, as an alchemist purifies the metal from other metals or impurities. In clinical specimens (sputum, stool, urine etc.), the mixed microorganisms are common, may mask and confuse the diagnostic results.

To avoid this confusion, a pure culture is needed. The extraneous organisms may change the pH, may damage or kill the desired microorganisms. A pure culture is the one which contains only one kind of microorganisms whereas a mixed culture contains two or more kinds of microorganisms.

Pure culture may be compared to a bed of roses obtained in pure culture of roses. The methods, used to isolate the bacteria in pure culture, consist of separating a single living bacterial cell and allowing it to multiply on a suitable culture medium, usually in a solid medium, to form a pure colony.

Bacteria multiply very rapidly, so that a single bacterial cell deposited on a solid medium gives rise to a mass which is called a colony.

1. Streaking or Plating (Inoculation of Culture Media):

If the material containing bacteria is streaked several times on the surface of a solid medium with a platinum loop, without recharging, fewer and fewer bacterial cells will remain on the loop as successive streaks are made and finally it may deposit a single bacterial cell on the surface. This procedure will thus give separate colonies of the organisms present in the material.

2. Dilution and Plating:

When the bacterial content of the material is expected to be high (e.g., feces), it is diluted with sterile nutrient broth or saline before plating.

3. Use of Selective Medium:

Media which promote the growth of one type of organisms and inhibit or retard the growth of others are known as “Selective Media”. Inhibiting substances like brilliant green, gentian violet, bile salts, potassium tellurite and penicillin are widely used in the preparation of such media.

4. Differential Sterilisation by Chemicals:

An example of this method is cultivation of Tubercle bacilli from sputum (which contains numerous other organisms) after preliminary treatment with dilute mineral acid or alkali. In Petroff’s method, the specimen of sputum is mixed with three or four times its volume of 4 per cent sodium hydroxide solution, incubated for one half an hour at 37°C and centrifuged.

The deposit is neutralized, in the presence of an indicator, by dilute hydrochloric acid and then seeded on a suitable medium. This treatment kills all other organisms except tubercle bacilli which are resistant to the action of dilute mineral acids, alkalis and other chemicals.”Antiformin” (Sodium hydroxide) can also be used instead of a dilute alkali.

5. Differential Sterilisation by Heat:

This method is employed where the organisms to be obtained in pure culture are more resistant to heat than others present in the material, e.g., separation of spore bearing bacilli (which are more resistant to heat) from the non-spore bearers. The material is heated at 60°C for 30 minutes, cooled and then inoculated in a suitable medium. Only the spore-bearers which have survived, will grow.

6. Inoculation of a Susceptible Animal:

This method is employed to isolate pathogenic microorganisms which are not easily grown on culture media or which are readily overgrown by the contaminating organisms. As example, we may quote isolation of tubercle bacilli from pus, peritoneal and pleural fluids by inoculating a guinea-pig and isolation of pneumococci by inoculating a mouse. In the latter case, the contaminating organisms are rapidly killed in the animal body whereas the pathogenic organisms multiply and can be recovered in pure culture from the tissues.

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